Ana Isa Pedroso Marcolino

UNIVERSIDADE FEDERAL DE SANTA MARIA CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS

Ana Isa Pedroso Marcolino

DESENVOLVIMENTO E AVALIAÇÃO DO POTENCIAL CITOTÓXICO DE COMPLEXOS DE INCLUSÃO DRONEDARONA/CICLODEXTRINAS

Santa Maria, RS 2017

Ana Isa Pedroso Marcolino

DESENVOLVIMENTO E AVALIAÇÃO DO POTENCIAL CITOTÓXICO DE

COMPLEXOS DE INCLUSÃO DRONEDARONA/CICLODEXTRINAS

Tese apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas, Área de Concentração I - Desenvolvimento e Avaliação de Produtos Farmacêuticos, da Universidade Federal de Santa Maria (UFSM, RS), como requisito parcial para obtenção do grau de Doutora em Ciências Farmacêuticas.

Orientadora: Prof. Dra. Clarice Madalena Bueno Rolim

Coorientadora: Prof. Dra. Daniele Rubert Nogueira Librelotto

Santa Maria, RS 2017

AGRADECIMENTOS

Agradeço a Deus, pelas bênçãos recebidas.

De modo especial, agradeço:

- a minha orientadora Prof. Dra. Clarice Rolim, pelas oportunidades de

trabalho e crescimento;

- a minha coorientadora Prof. Dra. Daniele Librelotto, cujo auxílio foi

fundamental para a realização do trabalho, pela amizade, apoio e incentivo;

- a Prof. Dra. Andréa Adams, pelo incentivo e apoio;

- aos meus colegas do Laboratório de Pesquisa em Avaliação

Biofarmacêutica e Controle de Qualidade (LABCQ) pela amizade, em especial a Ana

Christ, Juliana Santos, Karla Ribas, Laís Scheeren, Letícia Macedo, Mariane

Friedrich, Matheus Lago, Pauline Biscaino, Priscila Rosa e Suelen Burin;

- as queridas IC’s Joana Fernandes, Francieli Bellé e Josiele de Vargas pelo

auxílio na execução da parte experimental;

- a la Prof. Dra. María Pilar Vinardell por la oportunidad de realizar mi estancia

en su laboratório en la Facultad de Farmàcia de la Universitat de Barcelona;

- a la Prof. Dra. Montserrat Mitjans por su amistad y por enseñarme la técnica

del cultivo celular;

- a la Dra. Carmen Morán por su amistad y apoyo y a los compañeros del

grupo ITMC Lily Velazquez, Laura Marics, Marc Bilbao Asensio, Gloria Somalo

Barranco y Guillem;

- a minha querida mãe Jacira Pedroso por seu amor e apoio incondicionais;

- a minha família, especialmente a minha tia Leodovina Soares, pelas

orações;

- as minhas grandes amigas (irmãs) Isabella Trevisan, Laura Portes e Sabrina

Borin;

- ao CNPq e CAPES pelo auxílio financeiro;

- aos professores e funcionários do Departamento de Farmácia Industrial da

UFSM por auxiliarem indiretamente o desenvolvimento do trabalho.

BRANCO

Brancas substâncias

minuciosamente

medidas, pesadas

Alvos jalecos

mãos enluvadas

movimentam-se

pipetas, provetas

termômetros, alcoômetros

agitadores magnéticos

vidraria, HPLC

Brancas, as aspirações científicas

no cotidiano de mentes juvenis

perseguidoras incessantes

de significativos resultados

Sonhos muito brancos

aos céus, elevam-se

anjos da Ciência

abraçam-nos

sempre a postos e dispostos

a realizá-los...

Jacira Pedroso

RESUMO

DESENVOLVIMENTO E AVALIAÇÃO DO POTENCIAL CITOTÓXICO DE COMPLEXOS DE INCLUSÃO DRONEDARONA/CICLODEXTRINAS

AUTORA: Ana Isa Pedroso Marcolino

ORIENTADORA: Clarice Madalena Bueno Rolim COORIENTADORA: Daniele Rubert Nogueira Librelotto

A dronedarona é um novo agente antiarrítmico análogo à amiodarona. Foi aprovado para a manutenção do ritmo cardíaco normal em pacientes com fibrilação atrial. A dronedarona possui baixa biodisponibilidade e é instável no trato gastrintestinal. As ciclodextrinas são oligossacarídeos cíclicos com uma cavidade central relativamente hidrofóbica e superfície hidrofílica. Por formarem complexos com uma variedade de moléculas orgânicas, as ciclodextrinas têm sido amplamente utilizadas para aumentar a solubilidade, estabilidade e biodisponibilidade de fármacos pouco solúveis em água. No presente estudo, complexos de inclusão de dronedarona com β-ciclodextrina e 2-hidroxipropil-β-ciclodextrina foram preparados com o objetivo de melhorar a solubilidade aquosa e as propriedades de dissolução da dronedarona. Os complexos de inclusão no estado sólido foram obtidos pela mistura de quantidades de β-ciclodextrina e 2-hidroxipropil-β-ciclodextrina na proporção molar de 1:10 (fármaco:ciclodextrina). Os complexos foram preparados de acordo com os métodos de liofilização, coliofilização e malaxagem seguida de secagem por aspersão. Os estudos de solubilidade foram realizados pelo método do diagrama de solubilidade de fases. Os complexos no estado sólido foram caracterizados por calorimetria exploratória diferencial, difração de raios-X de pó, espectroscopia no infravermelho com transformada de Fourier e microscopia eletrônica de varredura. A caracterização dos complexos de inclusão por calorimetria exploratória diferencial e difração de raios-X mostrou que a dronedarona aparenta estar na forma amorfa. A dissolução dos complexos foi estudada e comparada com o fármaco puro. Após a complexação, houve um aumento significativo na porcentagem dissolvida da dronedarona em fluido gástrico simulado. A citotoxicidade dos complexos de inclusão foi avaliada em cultivo de fibroblastos da linhagem 3T3 utilizando o ensaio de redução do MTT (brometo 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2il-tetrazólico). Os complexos de inclusão com ambas as ciclodextrinas apresentaram uma significativa redução dos efeitos citotóxicos da dronedarona em comparação ao fármaco livre. Com a finalidade de determinar o potencial hepatotóxico da dronedarona livre e dos complexos de inclusão, avaliou-se a citotoxicidade dos compostos em células da linhagem HepG2, células tumorais de hepatoma humano. Nesse ensaio verificou-se um efeito dose-resposta, ou seja, o aumento da concentração dos compostos gerou uma redução da viabilidade celular. Não foi observada diferença significativa entre os valores de concentração inibitória (IC50) do fármaco livre e complexos de inclusão, sugerindo que a complexação do fármaco com ciclodextrinas não aumenta seu efeito hepatotóxico. O ensaio de fototoxicidade in vitro 3T3 NRU foi utilizado para verificar o potencial fototóxico e o fotoensaio utilizando células THP-1 e IL-8 foi usado para determinar o potencial fotossensibilizante do fármaco e dos complexos de inclusão de dronedarona com β-ciclodextrina e 2-hidroxipropil-β-ciclodextrina. O fármaco livre e os complexos de inclusão não apresentaram potencial fotoirritante. No ensaio de fotossensibilização, o complexo com β-CD obtido por malaxagem e secagem por aspersão mostrou potencial fotossensibilizante inferior ao do fármaco livre. Finalmente, as ciclodextrinas foram capazes de formar complexos com a dronedarona e desse modo, proporcionaram melhoria na solubilidade aquosa e estabilidade química do fármaco, além de reduzir seu potencial citotóxico. Assim, os complexos de inclusão demonstram ser uma alternativa promissora no âmbito farmacêutico, visando a obtenção de medicamentos com propriedades terapêuticas potencializadas.

Palavras-chave: Dronedarona. Antiarrítmico. Ciclodextrinas. Complexos de inclusão. Caracterização.

Citotoxicidade.

ABSTRACT

DEVELOPMENT AND EVALUATION OF THE POTENTIAL CYTOTOXICITY OF DRONEDARONE/CYCLODEXTRIN INCLUSION COMPLEXES

AUTHOR: Ana Isa Pedroso Marcolino ADVISER: Clarice Madalena Bueno Rolim

CO-ADVISER: Daniele Rubert Nogueira Librelotto

Dronedarone is a new antiarrhythmic agent, analogue of amiodarone. Dronedarone was approved for the maintenance of the sinus rhythmic in adult patients with atrial fibrillation. Dronedarone show bioavailability problems due to its very low water solubility, slow dissolution rate and instability in the gastrointestinal tract. Cyclodextrins are cyclic oligosaccharides with a relatively hydrophobic central cavity and a hydrophilic surface. Because cyclodextrins can form complexes with a variety of organic molecules, they have been widely used to increase the solubility, stability and bioavailability of poorly soluble drugs. In the present study, complexes of dronedarone with β-cyclodextrin (β-CD) and 2-hydroxypropyl- β-cyclodextrin (HP-β-CD) were prepared with the aim to increase the aqueous solubility and dissolution properties of dronedarone. Solid inclusion compounds were obtained by mixing appropriate amounts of dronedarone and β-CD or HP-β-CD, in a 1:10 molar ratio. The preparation was carried out according to the lyophilization, co-lyophilization and kneading and spray-drying methods. Solubility studies were performed by phase solubility analysis. The complexes were characterized in the solid state by DSC, XRD, FTIR spectroscopy and SEM. Characterization of inclusion complexes by DSC and XRD showed that dronedarone appeared to exist in a non-crystalline form. The solubility of the complexes were evaluated and compared with pure drug. Dronedarone solubility was notably improved in simulated gastric fluid. The cytotoxicity of the inclusion complexes was evaluated by a simple method based on 3T3 embryonic mouse fibroblast monolayers culture using the reduction of 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide (MTT) as in vitro viability assay. The inclusion complexes with both cyclodextrins produced a significant reduction in cytotoxic effects compared with the free dronedarone. In order to determine the hepatotoxic potential of the free drug and inclusion complexes, the cytotoxicity was investigated using human hepatoma cell line HepG2. The assay results showed a dose response effect; higher drug concentrations induced a higher reduction in cell viability. No significant difference among the IC50 values of the free drug and inclusion complexes was observed, suggesting that inclusion complexation did not increase dronedarone hepatotoxic effect. The 3T3 Neutral Red Uptake phototoxic test was used to verify the phototoxic potential, while the in vitro photoassay using THP-1 human monocytes, with the interleukin 8 (IL-8) expression as endpoint, was used to determine the photosensitizing potential of free dronedarone and its inclusion complexes with β-CD or HP-β-CD. The free drug and inclusion complexes did not show photoirritant potential. In the photosensitizing assay, inclusion complexes prepared with β-CD by kneading following spray-drying induced lower photosensitization in comparison to free dronedarone. Finally, cyclodextrins were able to form inclusion complexes with dronedarone, and provided an improved solubility and chemical stability, reducing drug cytotoxic potential. Thus, inclusion complexes with cyclodextrins might be a promising alternative in the development of formulations with improved therapeutic efficacy.

Keywords: Dronedarone. Antiarrhythmic drug. Cyclodextrins. Inclusion complexes. Characterization. Cytotoxicity.

LISTA DE ILUSTRAÇÕES

APRESENTAÇÃO Figura 1 - Estrutura química da amiodarona ............................................................. 29 Figura 2 - Estrutura química do cloridrato de dronedarona ....................................... 30 Figura 3 - Estrutura química da β-ciclodextrina (a) e representação esquemática da

estrutura tronco-cônica (b), respectivamente ....................................................... 36 Figura 4 - Diagrama de solubilidade de fases ........................................................... 40 ARTIGO 1 Figura 1 - Chromatograms of DRO tablet solution (A), reference solution (B) and

DRO inclusion complex with HP-β-CD prepared by lyophilization (C) at 20 μg mL -1 showing peak 1 = DRO; peaks 2,3,4 = degraded forms. (a) Non-degraded samples and samples submitted to stress degradation conditions such as: (b) alkaline hydrolysis with 1 M NaOH at 80°C for 0.5 h; (c) acidic hydrolysis with 3 M HCl at 80°C for 7 h and (d) after exposure to UVC light for 1.5 h. (D) UV reference solution spectchart obtained for the robustness assay of DRO in inclusion complex .................................. 64

Figura 2 - Pareto chart obtained for the robustness assay of DRO in inclusion complexhromatogram of DRO reference solution and DRO inclusion complex .................................................................................................... 68

Figura 3 - The full scan MS spectra (a-d) and the respective product ion spectra (e-h) of [M+H]+ of DRO reference substance (a) and degraded samples obtained under: (b) acidic hydrolysis; (c) alkaline hydrolysis and (d) after exposure to UV-C light.. ........................................................................... 70

Figura 4 - Plots of concentration (a) zero-order reaction, natural log of concentration (b) first-order reaction, and reciprocal of concentration (c) second-order reaction, against time, after the hydrolysis of DRO with 1.0 M NaOH at 60°C. ........................................................................................................ 72

Figura 5 - Cytotoxicity of DRO before and after degradation treatments (acidic, basic and photolytic stress conditions) on 3T3 cells as a function of concentration, as determined by MTT viability assay. Concentrations tested (from left to right) of 2.5 μg mL-1 (blank), 1.0 μg mL-1 (striped), 0.5 μg mL-1 (black) and 0.1 μg mL-1 (gray). The data represent the mean of three independent experiments ± SE (error bars). Statistical analyses were performed using ANOVA followed by Dunnett’s multiple comparison test. *

Statistically different (p < 0.05) and ** highly statistically different (p < 0.005) from non-degraded sample. Tukey’s multiple comparison test were also performed in order to verify if there is any difference on the cytotoxicity between the degradation times. However, no statistically significant differences were observed ....................................................... 73

ARTIGO 2 Figura 1 - Phase solubility diagrams of DRO with β-CD (blue) and HP-β-CD (red) in

aqueous solution at (a) 25 °C and (b) 37 °C. ......................................... 100 Figura 2 - DSC curves obtained for DRO (a), β-CD (b), HP-β-CD (c), physical mixture

with β-CD (d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with β-CD (f) and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by kneading following spray-drying with HP-β-CD (j) and β-CD (k) (10 °C.min-1 variations in temperature and in nitrogen atmosphere. .......................................................................................... 105

Figura 3 - PXRD patterns of DRO (a), β-CD (b), HP-β-CD (c), physical mixture with β-CD (d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with β-CD (f) and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by kneading following spray-drying with HP-β-CD (j) and β-CD (k). ................................................................................................... 111

Figura 4 - FT-IR spectra of DRO (a), β-CD (b), HP-β-CD (c), physical mixture with β-CD (d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with β-CD (f) and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by kneading following spray-drying with HP-β-CD (j) and β-CD (k). ................................................................................................... 117

Figura 5 - SEM micrographs of DRO (a), β-CD (b), HP-β-CD (c), physical mixtures with β-CD (d) and HP-β-CD (e), inclusion complexes obtained by colyophilization with β-CD (f) and HP-β-CD (g), by lyophilization with β-CD (h) and HP-β-CD (i), and by spray drying with β-CD (j) and HP-β-CD (k). ............................................................................................................... 121

Figura 6 - Dissolution profiles of free DRO and inclusion complexes obtained by lyophilization with β-CD (LB) and HP-β-CD (LH), by colyophilization with β-CD (RB) and HP-β-CD (RH), and by kneading and spray drying with β-CD (SB) and HP-β- CD (SH) in pH 1.2 (a), 4.5 (b) and 6.8 (c). .................... 124

Figura 7 – Cell viability of free DRO and inclusion complexes with β-CD and HP-β-CD obtained through different techniques on 3T3 cells, determined by the MTT assay. Assay concentrations (left to right): 1.25 µg mL-1 (dark gray), 2.5 µg mL-1 (light gray) and 5.0 µg mL-1. Statistical analyses were performed using ANOVA followed by Dunnett’s multiple comparison test. *

Statistically different (p < 0.05) using the free drug as control. .............. 127 Supplementary figure 1 - PXRD patterns of inclusion complexes obtained by lyophilization with β-CD (a), and HP-β-CD (b), by colyophilization with β-CD (c), and HP-β-CD (d) and by kneading following spray-drying with HP-β-CD (e) and β-CD (f) after storage in climate stability chamber for 30 days. ............................................ 137 Supplementary figure 2 - PXRD patterns of inclusion complexes obtained by lyophilization with β-CD (a), and HP-β-CD (b), by colyophilization with β-CD (c), and HP-β-CD (d) and by kneading following spray-drying with HP-β-CD (e) and β-CD (f) after storage into desiccator for 30 days. ................................................................ 138 ARTIGO 3 Figura 1 - Dose response curves of DRO (A) and inclusion complexes prepared by

colyophilization with β-CD (B) and HP-β-CD (C), by lyophilization with β-CD (D) and HP-β-CD (E) and by kneading following spray-drying with β-CD (F) and HP-β-CD (G) in non-irradiated (diamonds) and irradiated (squares) in NIH-3T3 cells. Results are presented as mean ± SE of three independent experiments, and statistical analysis was performed with Dunnett’s multiple comparison test (*p < 0.05). ...................................... 159

Figura 2 – Cytotoxicity rates measure by the MTT assay for non-irradiated (gray) and irradiated (black) conditions. The concentration tested for dronedarone (D) and inclusion complexes (LH, RH, SH, SB, RB, LB) were 2.5 µg/mL (a), 1.25 µg/mL (b) and 0.625 µg/mL (c). ..................................................... 162

Figura 3 – IL-8 release induced by increasing concentrations of free DRO (a), inclusion complexes prepared by colyophilization with β-CD (b) and HP-β-CD (c), by lyophilization with β-CD (d) and HP-β-CD (e), and by kneading following spray-drying with β-CD (f) and HP-β-CD (g), and chlorpromazine (h) in non-irradiated (open circles) and irradiated cells (black squares). SI

calculated for each concentration tested is also shown (black triangles). Results are presented as mean ± S.E.M., and statistical analysis was performed with two-sample t-test. (*p < 0.05; **p < 0.01). ..................... 163

Figura 4 – Effects of DRO and inclusion complexes on IL-8 release. THP-1 cells (irradiated and non-irradiated) were treated with the compounds at a concentration of 1.25 µg/mL for 24 h. IL-8 release was measured by ELISA in culture supernatants, results expressed in pg/mL, representing the mean ± S.E.M. Statistical analysis was performed with two-sample t-test, with *p< 0.05 versus DRO. ............................................................. 164

Figura 5 –The increases of IL-8 release expressed as stimulation indexes for non-irradiated (NI-SI) and irradiated cells (I-SI). An overall stimulation index (I-SI/NI-SI) was calculated as the ratio of the stimulation indexes in irradiated and non-irradiated cells. The concentrations assayed were: chlorpromazine (CPZ) 0.1 µg/mL, DRO 1.25 µg/mL and inclusion complexes (RB, RH, LB, LH, SB and SH) equivalent to 1.25 µg/mL of DRO. .................................................................................................... 164

Figura 6 – Concentration response curve from 24 h-exposure of HepG2 cells to free DRO. Data are expressed as mean ± S.E.M. of three independent experiments performed in triplicate. Statistical analysis was performed using two- sample t-test. *p<0.05 versus control, **p<0.001 versus controle, ***p<0.00001 versus controle, p<0.05 versus 7.50 µg/mL. ... 167

Figura 7 -Cytotoxicity of free DRO and inclusion complexes prepared by colyophilization with β-CD (RB) and HP-β-CD (RH), by lyophilization with β-CD (LB) and HP-β-CD (LH) and by kneading following spray-drying with β-CD (SB) and HP-β-CD (SH) expressed as IC50 values (µg/mL) in HepG2 cells measured by the MTT assay. Data represent the mean ± S.E.M. of three independent experiments. ........................................... 168

LISTA DE TABELAS

APRESENTAÇÃO Tabela 1 - Propriedades físico-químicas das ciclodextrinas ...................................... 36 Tabela 2 – Métodos analíticos disponíveis na literatura para determinação de dronedarona .............................................................................................................. 44 ARTIGO 1

Table 1 - Amount of DRO degraded in each stress condition for tablet, reference and inclusion complex solutions. .............................................................. 65

Table 2 - Intra-day and inter-day precision data for the proposed HPLC method .... 66 Table 3 - Recovery studies for the HPLC method ................................................... 67 Table 4 - The robustness testing of the HPLC method for DRO in tablets. ............. 68 ARTIGO 2 Table 1 - Results of DRO intrinsic solubility (S0), maximum solubility (Smax), and

solubility efficiency (SE), slope and stability (KC) and complexation efficiency (CE) constants, from phase solubility diagrams at 25°C. ....... 101

Table 2 - Drug content of inclusion complexes of DRO with β-CD and HP-β-CD obtained by the HPLC method and yield of each preparation technique. .............................................................................................................. 103

Table 3 - -Thermal analyses obtained by DSC for dronedarone, β-CD, HP-β-CD, physical mixtures and inclusion complexes prepared by different techniques. ............................................................................................ 109

Table 4 - -Thermal analysis by TGA to β-CD, HP-β-CD and DRO. ...................... 109

LISTA DE ABREVIATURAS

ANVISA Agência Nacional de Vigilância Sanitária

β-CD Betaciclodextrina

CD Ciclodextrina

CLAE Cromatografia líquida de alta eficiência

CYP Citocromo P450

DAD Detector de arranjo de diodos

DMSO Dimetilsulfóxido

DPR Desvio padrão relativo

DRO Dronedarona

DRXP Difração de raios-X de pó

DSC Calorimetria exploratória diferencial

EMA European Medicines Agency

FA Fibrilação atrial

FDA U. S. Food and Drug Administration

FBS Soro fetal bovino

HP-β CD 2-hidroxipropil-β-ciclodextrina

ICH International Conference on Harmonisation

IV Infravermelho

LB Complexo de inclusão com β-CD obtido pela técnica de liofilização

LH Complexo de inclusão com HP-β-CD obtido pela técnica de liofilização

MEV Microscopia eletrônica de varredura

MTT Brometo 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2il-tetrazólico

r Coeficiente de correlação

RB Complexo de inclusão com β-CD obtido pela técnica de coliofilização

RH Complexo de inclusão com HP-β-CD obtido pela técnica de coliofilização

SB Complexo de inclusão com β-CD obtido pela técnica de malaxagem seguido de secagem por aspersão

SH Complexo de inclusão com HP-β-CD obtido pela técnica de malaxagem seguido de secagem por aspersão

SUMÁRIO 1 APRESENTAÇÃO ................................................................................................. 24 1.2 REFERENCIAL TEÓRICO .................................................................................. 28 1.3 PROPOSIÇÃO .................................................................................................... 50 2 ARTIGO 1 – CINÉTICA DE DEGRADAÇÃO, ESTUDOS DE CITOTOXICIDADE IN VITRO E VALIDAÇÃO DE MÉTODO POR CLAE INDICATIVO DE ESTABILIDADE PARA CLORIDRATO DE DRONEDARONA EM COMPRIMIDOS E EM COMPLEXOS DE INCLUSÃO COM CICLODEXTRINAS ........................................ 52 3 ARTIGO 2 - PREPARAÇÃO, CARACTERIZAÇÃO E ESTUDO DE CITOTOXICIDADE DE COMPLEXOS DE INCLUSÃO DE DRONEDARONA E CICLODEXTRINAS .................................................................................................. 82 4 ARTIGO 3 - AVALIAÇÃO DO POTENCIAL HEPATOTÓXICO, FOTOTÓXICO E FOTOSSENSIBILIZANTE DO CLORIDRATO DE DRONEDARONA E SEUS COMPLEXOS DE INCLUSÃO COM CICLODEXTRINAS ..................................... 140 5 DISCUSSÃO ...................................................................................................... 172 6 CONCLUSÃO .................................................................................................... 180 REFERÊNCIAS ...................................................................................................... 184

1 APRESENTAÇÃO

26

1 APRESENTAÇÃO

A fibrilação atrial (FA) é a arritmia cardíaca mais comumente encontrada em

pacientes idosos. Essa arritmia, resultante de etiologia cardíaca e não cardíaca, é

um potente fator de risco para comorbidades como o acidente vascular cerebral,

além de estar associada a altos custos para o sistema de saúde (GO; HYLEK;

PHILLIPS, 2001; NATTEL, 2002; YALTA et al., 2009).

A dronedarona é um novo agente antiarrítmico desenvolvido para o

tratamento de pacientes com FA. Em 2009, foi aprovada pelo U.S. Food and Drug

Administration (FDA), pela European Medicines Agency (EMA) e pela Agência

Nacional de Vigilância Sanitária (ANVISA) para reduzir o risco de hospitalização por

arritmia nesses pacientes (BRASIL, 2009; EMA, 2012; U.S. FOOD AND DRUG

ADMINISTRATION, 2014). Está disponível comercialmente na forma farmacêutica

de comprimidos revestidos.

A baixa solubilidade em água da dronedarona (0,64 mg/mL), associada ao

metabolismo de primeira passagem, conduz a uma biodisponibilidade absoluta

prejudicada (aproximadamente 15%), sendo necessária uma elevada quantidade de

fármaco (400 mg administrados duas vezes ao dia) para a obtenção do efeito

terapêutico adequado (AUSPAR, 2010; EMA, 2012; U.S. FOOD AND DRUG

ADMINISTRATION, 2014).

Quando um fármaco é administrado no estado sólido, este precisa estar

inicialmente dissolvido nos fluidos corporais, a fim de permear as barreiras, como a

mucosa do trato gastrintestinal para posteriormente alcançar o sítio de ação e tornar-

se terapeuticamente efetivo. As partículas do fármaco no estado sólido (na forma

cristalina ou amorfa) devem se dissolver de forma apropriada após a administração,

a fim de serem transportadas adequadamente até o sítio alvo. Nesse sentido,

aumentar a solubilidade aquosa de fármacos pouco solúveis em água, para

fármacos nos quais a dissolução é o fator limitante da absorção, pode aumentar a

fração de fármaco absorvida (LOFTSSON; MUELLERTZ; SIEPMANN, 2013). Uma

estratégia para melhorar a solubilidade de fármacos é a formação de complexos de

inclusão com ciclodextrinas. Esse excipiente funcional tem sido utilizado

principalmente para solubilizar fármacos pouco solúveis em água, no aumento da

sua biodisponibilidade oral e da estabilidade de medicamentos e na redução de seus

efeitos colaterais (OLIVEIRA; SANTOS; COELHO, 2009). As ciclodextrinas são

27

moléculas que apresentam uma estrutura tronco-cônica, que se caracteriza pela

presença de cavidade de natureza apolar. Essa cavidade pode acomodar moléculas

no seu interior, sem que haja o estabelecimento de ligações covalentes entre as

duas entidades, constituindo essa a principal base de sua utilização farmacêutica

(LOFTSSON; DUCHÊNE, 2007; SA BARRETO; CUNHA-FILHO, 2008). A

betaciclodextrina (β-CD) é a ciclodextrina mais empregada em formulações

farmacêuticas devido ao seu baixo custo. Entretanto, sua solubilidade aquosa é

limitada e considerando sua toxicidade, foi desenvolvido um derivado hidrofílico, a 2-

hidroxipropil-betaciclodextrina (HP-β-CD), que pode ser administrada por via

intravenosa e atualmente é comercializada em muitos produtos farmacêuticos

aprovados pelo FDA (JAMBHEKAR; BREEN, 2016).

Nesse sentido, visando o aumento da solubilidade do fármaco, foram

desenvolvidos complexos de inclusão de dronedarona com β-CD e HP-β-CD, que

foram caracterizados através de técnicas físico-químicas. Além disso, a

citotoxicidade in vitro da dronedarona e dos complexos de inclusão em cultura de

fibroblastos da linhagem 3T3 foi avaliada por meio do ensaio colorimétrico MTT.

Posteriormente, a hepatotoxicidade, o potencial fototóxico e fotossensibilizante da

dronedarona livre e dos complexos de inclusão foram investigados utilizando

modelos celulares in vitro, a fim de investigar possíveis alterações na citotoxicidade

do fármaco em função da complexação com ciclodextrinas.

28

1.2 REFERENCIAL TEÓRICO

1.2.1 Tratamento da fibrilação atrial

A fibrilação atrial (FA) é a perturbação do ritmo cardíaco mais encontrada na

prática clínica, sendo responsável por cerca de um terço das hospitalizações devido

a distúrbios cardíacos (GO; HYLEK; PHILLIPS, 2001). Essa disfunção é um dos

principais determinantes de acidente vascular cerebral e pode reduzir a qualidade de

vida e o desempenho cardíaco, além de estar associada com o aumento da

mortalidade em pacientes com insuficiência cardíaca (NATTEL, 2002). A FA pode

ser definida como “uma taquiarritmia supraventricular caracterizada pela ativação

atrial descoordenada com consequente deterioração da função mecânica atrial”

(FUSTER et al., 2006). A prevalência da FA aumenta com a idade e altas taxas são

encontradas na população idosa. No ano de 2010, o número global estimado de

indivíduos com FA foi de 33,5 milhões (20,9 milhões de homens e 12,6 milhões de

mulheres). Em relação ao ônus associado a FA, medido como anos de vida perdidos

por incapacidade, houve um aumento de 18,8% para os homens e 18,9% para as

mulheres entre os anos de 1990 e 2010 (CHUGH et. al., 2014). As projeções para o

ano de 2030 estimam que o número de pacientes com FA esteja entre 14-17

milhões, e o número de novos casos por ano seja entre 120.000 a 215.000 na

Europa (ZONI-BERISSO et. al., 2014), justificando-se a necessidade de estratégias

para a prevenção e tratamento da FA.

O maior objetivo terapêutico para os pacientes com FA é a restauração e

manutenção do ritmo cardíaco normal. Devido à alta taxa de recorrência da FA, há a

necessidade de um tratamento contínuo com antiarrítmicos (SINGH; ALIOT, 2007).

Os fármacos antiarrítmicos utilizados no tratamento da FA são geralmente

classificados em quatro categorias baseadas em suas propriedades

eletrofisiológicas: bloqueadores de canais de sódio (classe I), bloqueadores dos

receptores beta-adrenérgicos (classe II), bloqueadores de canais de potássio (classe

III) e bloqueadores de canais de cálcio (classe IV) (BRUNTON; LAZO; PARKER,

2006).

A amiodarona (Figura 1) foi reconhecida primeiramente por prolongar a

duração do potencial cardíaco após tratamento contínuo e, assim, foi classificada

como agente classe III (VARRÓ et al., 2001). Esse agente antiarrítmico é efetivo no

29

tratamento de arritmias ventriculares e supraventriculares e na prevenção de morte

por cardiomiopatia não isquêmica. Entretanto, a amiodarona está associada a

efeitos adversos significativos como toxicidade pulmonar, hepática, no sistema

nervoso periférico, ocular, cutânea e disfunção tireoidiana (SINGH; ALIOT, 2007).

Os efeitos deletérios têm sido relacionados à presença do iodo no anel aromático,

que torna a molécula mais lipofílica e aumenta a sua distribuição em locais do

organismo como a tireoide, pulmões, fígado, córnea, pele e nervos periféricos

(LAUGHLIN; KOWEY, 2008).

Figura 1 – Estrutura química da amiodarona.

A dronedarona foi desenvolvida como uma alternativa à amiodarona, com

atividade antiarrítmica semelhante, porém com menor toxicidade. No

desenvolvimento da molécula da dronedarona, os átomos de iodo foram eliminados

e um grupo metanosulfonil foi adicionado ao grupamento benzofurano. As alterações

estruturais reduziram a lipofilicidade da dronedarona e consequentemente, a sua

meia-vida (para aproximadamente 24h), além de reduzir a acumulação tecidual.

Essas alterações foram realizadas com o intuito de reduzir a toxicidade pulmonar e

tireoidiana relacionada com a amiodarona (HOHNLOSER et al., 2009; SUN;

SARMA; SINGH, 2002).

1.2.2 Dronedarona

1.2.2.1 Características físico-químicas

O cloridrato de dronedarona (SR33589B, Figura 2) é um derivado

benzofurano estruturalmente relacionado com a amiodarona, denominado

30

quimicamente cloridrato de N-{2-butil-3-[4-(3-dibutil-aminopropóxi) benzoil]

benzofurano-5-il} metanosulfonamida. É um pó fino branco, praticamente insolúvel

em água e em alguns pHs fisiológicos e livremente solúvel em cloreto de metileno e

metanol. Na literatura, a solubilidade descrita é 0,64 mg/mL em água e 0,01 mg/mL

em pH 1,2 e em tampão fosfato pH 7,0 (AusPAR, 2010). A solubilidade em meio

fracamente ácido (pH 3-5) é de aproximadamente 1 a 2 mg/mL, sendo, portanto, pH-

dependente (HAN et al., 2015a). A fórmula empírica é C31H44N2O5S, HCl com massa

molecular de 593,2 g/mol (U.S. FOOD AND DRUG ADMINISTRATION, 2014). A

dronedarona possui grupo nitrogênio terciário em sua estrutura e seu valor de pKa

calculado é 9,4. O valor de log P calculado é 6,36 (XIE et al., 2011).

Figura 2 – Estrutura química do cloridrato de dronedarona.

1.2.2.2 Propriedades eletrofisiológicas da dronedarona e farmacocinética

Em modelos animais, a dronedarona previne a ocorrência de taquicardia e

fibrilação ventricular e restaura o ritmo cardíaco normal, devido a seus efeitos

eletrofisiológicos semelhantes à amiodarona (VARRÓ et al., 2001; SUN; SARMA;

SINGH, 2002; GAUTIER et al., 2003). Esses efeitos se devem as suas propriedades

eletrofisiológicas de um agente antiarrítmico de classe III, embora demonstre

atividade eletrofisiológica de várias classes (GAUTIER et al., 2003; EMA, 2012). Por

ser um bloqueador multicanal, a dronedarona prolonga o potencial de ação cardíaco

e os períodos refratários (curto período de tempo em que a célula não pode ser

reestimulada) através da inibição das correntes de sódio, potássio e cálcio, incluindo

as correntes de potássio regeneradoras rápidas e a corrente de cálcio do tipo L

(VARRÓ et al., 2001; SUN; SARMA; SINGH, 2002; GAUTIER et al., 2003; LALEVÉE

et al., 2003; WATANABE; KIMURA, 2008).

31

A dronedarona é bem absorvida (~70% a 94%) após administração oral dos

comprimidos contendo 400 mg quando a administração é concomitante com uma

refeição rica em gorduras, que aumenta a absorção de 2 a 3 vezes (PATEL; YAN;

KOWEY, 2009). Assim, é afetada significativamente pela ingestão de alimentos. Sua

biodisponibilidade absoluta é de 4% sem a presença de alimentos, que aumenta

para 15% com uma dieta rica em lipídeos, entretanto esses baixos valores se devem

ao efeito de primeira passagem associado ao seu metabolismo pré-sistêmico (BIN

JARDAN; GABR; BROCKS, 2014; U.S. FOOD AND DRUG ADMINISTRATION,

2014). Após a administração oral no estado alimentado, as concentrações

plasmáticas máximas da dronedarona e de seu metabólito ativo N-

debutildronedarona são alcançadas entre 3 e 6 horas. Após a administração repetida

de 400 mg duas vezes ao dia juntamente com a refeição, o estado de equilíbrio é

alcançado dentro de 4 a 8 dias de tratamento, com a mediana da Cmax da

dronedarona de 84-147 ng/mL. Acima de 80% da dose oral é excretada nas fezes,

principalmente na forma de metabólito e menos de 6% é recuperada na urina,

também em sua maior parte como metabólito (BIN JARDAN; BROCKS, 2016). A

dronedarona possui meia-vida de eliminação terminal de aproximadamente 25 a 30

horas, sendo a do seu metabólito N-debutil de aproximadamente 20 a 25 horas

(YALTA et al., 2009; EMA, 2012; U.S. FOOD AND DRUG ADMINISTRATION, 2014;

HAN et al., 2015).

A dronedarona é um substrato e inibidor moderado do CYP3A4 e um fraco

inibidor do CYP2D6 (CHENG, 2010). Estudos de interações medicamentosas

demonstraram que a dronedarona afeta a farmacocinética do carvedilol através da

inibição da atividade hepática do CYP2D6 (KIM; BAEK, 2018). A dronedarona tem o

potencial para inibir o sistema de efluxo da glicoproteína-P (P-gP), aumentando a

exposição a substratos da P-gP como a digoxina, quando da administração

concomitante (HOY; KEAM, 2009).

O perfil farmacocinético da dronedarona após administração oral e

intraperitoneal em ratos e o efeito da hiperlipidemia foram estudados por Bin Jardan

e Brocks (2016). Os resultados do estudo demonstraram um alto volume de

distribuição no rato e alta ligação às proteínas plasmáticas, tanto no plasma

normolipidêmico como no hiperlipidêmico. O fármaco apresentou baixa

biodisponibilidade (<20%) após ambas as vias de administração. As elevadas

concentrações plasmáticas após a administração oral a ratos hiperlipidêmicos foi

32

atribuída a um efeito direto nas enzimas metabolizadoras ou nas proteínas de

transporte.

A dronedarona foi aprovada em 2009 pelo FDA (PAGE; HAMAD;

KIRKPATRICK, 2009; U.S. FOOD AND DRUG ADMINISTRATION, 2014), indicada

para reduzir o risco de hospitalização relacionada à FA, em pacientes com ritmo

cardíaco normal e com histórico de FA paroxística ou permanente. De acordo com a

European Medicines Agency, é indicada para manutenção do ritmo cardíaco normal

após cardioversão elétrica em pacientes adultos clinicamente estáveis com presença

atual de FA não permanente (EMA, 2012). Em relação aos seus efeitos adversos

relatados na literatura, destaca-se a hepatotoxicidade (U.S. FOOD AND DRUG

ADMINISTRATION, 2014) e reação de fotossensibilidade após a administração do

fármaco (KUO; MENON; KUNDU, 2014).

1.2.2.3 Forma farmacêutica comercial

O fármaco está disponível comercialmente na União Europeia e em alguns

países como Estados Unidos, Canadá, Austrália, Índia e Cingapura na forma

farmacêutica de comprimidos revestidos contendo 400 mg de dronedarona (na

forma de cloridrato) (Multaq®, Sanofi, França). Os excipientes declarados são:

- Núcleo dos comprimidos: hidroxipropilmetilcelulose, amido de milho,

crospovidona, poloxâmero 407, lactose mono-hidratada, sílica coloidal anidra,

estearato de magnésio.

- Revestimento/polimento dos comprimidos: hidroxipropilmetilcelulose,

polietilenoglicol 6000, dióxido de titânio, cera de carnaúba (U.S. FOOD AND DRUG

ADMINISTRATION, 2014).

Em relação ao revestimento dos comprimidos, a hidroxipropilmetilcelulose é

um polímero formador de filme de caráter hidrofílico, que por possuir a capacidade

de intumescimento/relaxamento, pode ser utilizado para controlar a liberação de

fármacos a partir da matriz em sistemas de liberação modificada (ROLIM et al.,

2009). A cera de carnaúba é um agente de revestimento utilizado isolado ou em

conjunto com outros excipientes como a hidroxipropilmetilcelulose, para produzir

formas farmacêuticas sólidas de liberação controlada (ROWE; SHESKEY; OWEN,

2006). O polietilenoglicol pode ser empregado como plastificante, para melhorar a

qualidade dos filmes de revestimento (ROLIM et al., 2009). Além disso, o

33

polietilenoglicol pode melhorar a solubilidade aquosa e conferir permeabilidade ao

filme, para garantir a penetração pelos fluidos biológicos, melhorando assim a

dissolução de compostos pouco solúveis em água (ALLEN; POPOVICH; ANSEL,

2007).

O fabricante original desenvolveu uma formulação baseada em uma

dispersão sólida contendo o agente solubilizante poloxâmero 407, com a finalidade

de aumentar a dissolução no trato gastrintestinal e, assim, aumentar

significativamente a sua biodisponibilidade no estado de jejum. O fabricante afirma

que o aumento da biodisponibilidade da dronedarona causado pelo poloxâmero na

formulação é devido ao aumento da solubilidade. Entretanto, dados da literatura

sugerem que o poloxâmero possui ação inibitória da P-gP, que pode ser um fator

contribuinte (EMA, 2012). Nesta dispersão sólida preparada pelo método do

solvente, o fármaco pode estar disperso molecularmente no carreador hidrofílico,

formando uma estrutura amorfa. Assim, a formulação apresenta uma maior

superfície, que aumenta a velocidade de dissolução da molécula pouco solúvel em

água (HAN et al., 2015a).

1.2.2.4 Sistemas de liberação contendo dronedarona

Veena e col. (2013) prepararam péletes microporosos carregados com

dronedarona formados por mistura (blenda) de celulose microcristalina com cloreto

de sódio, através da técnica de extrusão/ esferonização. Os péletes foram

caracterizados por microscopia eletrônica de varredura, que confirmou sua

morfologia porosa. A análise da formulação por espectroscopia no infravermelho

revelou a presença de bandas características da dronedarona no espectro, e a

análise por calorimetria exploratória diferencial indicou ponto de fusão próximo ao

ponto de fusão do fármaco puro, sugerindo a ausência de interação entre o fármaco

e o polímero. Estudos de liberação foram realizados em meios ácido (pH 1,2) e

alcalino (pH 7,4), demonstrando que o aumento da concentração de polímero nos

péletes reduziu a liberação do fármaco, devido a hidrofobicidade do polímero.

Ressalta-se que não houve liberação significativa do fármaco em pH gástrico. Já em

pH alcalino, a liberação do fármaco a partir dos péletes variou entre 73 a 92%, que

pode ser associada a diferente concentração de polímero empregada nas

formulações avaliadas.

34

Com a finalidade de aumentar a velocidade de dissolução do fármaco, Han e

col. (HAN et al., 2015a, 2015b) desenvolveram uma dispersão sólida preparada por

extrusão por fusão com um solubilizante polimérico (Soluplus®). As dispersões

sólidas foram caracterizadas quanto ao teor de fármaco e caracterizadas por

difração de raios-X e microscopia eletrônica de varredura. Os difratogramas

mostraram uma amorfização do fármaco nas dispersões sólidas, em comparação

com a mistura física fármaco-polímero e as micrografias demonstraram que o

fármaco estava incorporado ao carreador polimérico. Além disso, foram preparados

comprimidos com as dispersões sólidas, que juntamente com as últimas e com o

fármaco puro, foram submetidos ao teste de dissolução na faixa de pH de 1,2 a 6,8.

A quantidade cumulativa de fármaco liberada em 120 min a partir das dispersões

sólidas foi de aproximadamente 80% em todos os meios testados, e foi superior ao

fármaco livre nos meios com pH 1,2 e 6,8 (HAN, 2015a). Os mesmos autores

também desenvolveram um sistema de liberação de fármacos auto-

microemulsificante, a fim de reduzir a interação medicamento-alimento (food effect).

A formulação lipídica consistiu em dronedarona base (não na forma de cloridrato)

dissolvida em um pré-concentrado, com o solubilizante Labrafil M 1944CS (oleil-6

glicerídeo de polietilenoglicol) e Kolliphor EL (óleo de rícino polietoxilado). A

formulação foi comparada ao produto comercial (Multaq®) quanto ao perfil de

dissolução e ao perfil farmacocinético após administração oral em cães da raça

beagle. A taxa de dissolução do fármaco a partir da formulação foi significativamente

maior, em torno de 80% de liberação em 60 min, em relação ao produto comercial,

principalmente nos pHs 1,2 e 6,8. No perfil farmacocinético, a interação fármaco-

alimento foi menor no grupo da formulação, entretanto não houve diferença

significativa nas concentrações do estado alimentado. Já no estado de jejum, a área

sob a curva e concentração máxima foram em torno de três vezes maiores para a

formulação em comparação aos comprimidos comerciais (HAN, 2015b).

Nanopartículas contendo cloridrato de dronedarona foram preparadas pela

técnica da precipitação com anti-solvente, utilizando goma de Caesalpinia pulcherrima

como estabilizante (YEOLE et al., 2016). A morfologia das nanopartículas foi

avaliada por microscopia eletrônica de varredura, que mostrou agregados de

nanopartículas na forma esférica, com tamanho entre 300 e 600 nm. O espectro no

infravermelho (FT-IR) das nanopartículas demonstrou ser idêntico ao espectro do

fármaco livre, sugerindo que não houve alteração da estrutura química do fármaco

35

após o processo de precipitação. As nanopartículas também foram examinadas por

calorimetria exploratória diferencial e o ponto de fusão detectado foi inferior

(141,55°C) ao ponto de fusão do fármaco puro (146,55°C). Os autores afirmam que

a diferença se deve a redução do tamanho de partícula do fármaco para a escala

nanométrica e redução da cristalinidade devido ao rápido processo de nucleação,

resultando em um cristal imperfeito. As análises por difração de raios-X indicaram

que o fármaco presente nas nanopartículas se encontra no estado cristalino, com

uma leve redução da cristalinidade, evidenciada pela redução na intensidade dos

picos característicos da dronedarona. A solubilidade das nanopartículas em pH 4,5

foi ligeiramente superior ao fármaco puro, possivelmente devido a redução do

tamanho de partícula. O mesmo efeito foi evidenciado nos estudos de dissolução em

tampão fosfato pH 4,5, com aumento na velocidade de dissolução, favorecida

também pelo aumento da área superficial, pela presença do agente estabilizante

hidrofílico nas nanopartículas e pela redução da cristalinidade do fármaco.

1.2.3 Ciclodextrinas e complexos de inclusão

1.2.3.1 Ciclodextrinas

As ciclodextrinas (CD) são oligossacarídeos cíclicos, constituídos por um

número variável de unidades de D-glicose, obtidas a partir da degradação

enzimática do amido pela enzima ciclodextrina-α-glicosil-transferase (CGtase). As

ciclodextrinas naturais mais comuns apresentam seis, sete ou oito unidades de D-

glicopiranose unidas por ligações α (1,4) e são denominadas α-, β-, γ-ciclodextrinas,

respectivamente (SA BARRETO; CUNHA-FILHO, 2008).

A estrutura molecular das ciclodextrinas apresenta a forma tronco-cônica com

propriedades únicas (Figura 3). Essa forma é devido à ausência de livre rotação das

ligações glicosídicas e da conformação em cadeira das moléculas de glicose. Nessa

conformação, todos os grupos hidrofílicos estão orientados para o exterior da

molécula, conferindo-lhe um caráter hidrofílico e promovendo a sua solubilização em

meio aquoso. A cavidade apresenta natureza hidrofóbica devido à formação de dois

anéis de grupos C-H e de um anel composto por átomos de oxigênio incluídos nas

ligações glicosídicas (JAMBHEKAR, BREEN, 2016).

36

Figura 3 – Estrutura química da β-ciclodextrina (a) e representação esquemática da estrutura tronco-cônica (b), respectivamente.

Fonte: (JAMBHEKAR, BREEN, 2016).

A β-CD é a ciclodextrina mais empregada em formulações farmacêuticas. Isso

se deve a sua fácil produção e consequentemente baixo custo, seu preço médio é

de aproximadamente 5 dólares/kg. Entretanto, sua solubilidade aquosa é limitada,e

por isso é inadequada para administração parenteral. Essa limitação impulsionou o

desenvolvimento de derivados da β-CD, através da substituição das múltiplas

hidroxilas da β-CD em ambos os anéis da molécula. A 2-hidroxipropil-β-ciclodextrina

(HP-β-CD) é um derivado da β-CD e pertence ao grupo das ciclodextrinas

hidroxialquiladas. É obtida por condensação do óxido de propileno com a β-

ciclodextrina. A solubilidade aquosa dos derivados é superior a solubilidade das

ciclodextrinas naturais (Tabela 1), pois as últimas se apresentam no estado

cristalino, caracterizado por fortes ligações de hidrogênio entre as moléculas,

enquanto que os derivados apresentam uma redução da cristalinidade (a ligação de

cadeias orgânicas causa a quebra das ligações de hidrogênio intermoleculares

(LOFTSSON; DUCHÊNE, 2007; KURKOV, LOFTSSON, 2013).

Tabela 1 – Propriedades físico-químicas das ciclodextrinas

Propriedade β-CD HP-β-CD

Massa molecular (g/mol) 1135 1400a Solubilidade em água (mg/ mL) 25°C 18,5 >600 Faixa de fusão (°C) 255-265 - pKa a 25°C 12,2 -

aValor informado pelo fornecedor ou calculado de acordo com o grau de substituição.

Fonte:(MURA, 2014; JAMBHEKAR; BREEN, 2016; IACOVINO et. al., 2017).

37

Dentre os excipientes farmacêuticos, as CD tem um perfil toxicológico mais

favorável em relação a surfactantes, polímeros solúveis em água e solventes

orgânicos. Devido a sua origem, a partir de degradação enzimática do amido, e sua

característica hidrofílica, considerando o elevado número de hidrogênios doadores e

receptores, sua biodisponibilidade oral é muito baixa (abaixo de 4%) e, portanto atua

como um carreador. Após administração oral, as CDs são muito pouco absorvidas

na circulação sistêmica e são metabolizadas pelo trato gastrintestinal principalmente

por digestão bacteriana, formando oligossacarídeos, monossacarídeos e gases

como hidrogênio, dióxido de carbono e metano. Os derivados das CDs, como a HP-

β-CD, possuem um perfil toxicológico melhorado em relação as CDs naturais de

origem e por isso têm sido amplamente utilizados em formulações injetáveis. Após

administração parenteral, as ciclodextrinas hidrofílicas são excretadas inalteradas

por via renal com depuração plasmática total próxima a taxa de filtração glomerular

(LOTFSSON; BREWSTER, 2012; LOFTSSON et. al., 2016).

As ciclodextrinas são utilizadas em inúmeras áreas, incluindo a indústria

farmacêutica e cosmética, agroquímica, alimentar, entre outras. O uso de

ciclodextrinas em formulações farmacêuticas deve-se, sobretudo, às suas

propriedades de complexação, permitindo aumentar a solubilidade aquosa de

fármacos lipofílicos, sua estabilidade e biodisponibilidade, quando a solubilidade e a

dissolução são fatores limitantes na liberação do fármaco. Ciclodextrinas amorfas

como a HP-β-CD são úteis para a inibição da transição polimórfica e taxa de

cristalização de fármacos pouco solúveis em água durante o armazenamento, que

pode consequentemente manter as características de elevada dissolução e

biodisponibilidade oral dos fármacos (UEKAMA, 2004; LOFTSSON; DUCHÊNE,

2007).

Além disso, as ciclodextrinas podem ser utilizadas para mascarar odores e

sabor desagradáveis de certos fármacos, na prevenção de interações e

incompatibilidades e na conversão de fármacos líquidos em produtos sólidos. O

aumento da atividade do fármaco e a redução de seus efeitos colaterais podem ser

obtidos através da formação de complexos de inclusão. Esse grupo de excipientes

farmacêuticos úteis e seus complexos de inclusão podem ser utilizados na

preparação de formas farmacêuticas sólidas, líquidas e semissólidas com aplicação

nas vias de administração oral, parenteral, pulmonar, nasal, bucal, sublingual, retal,

ocular e dérmica (LOFTSSON; DUCHÊNE, 2007; SA BARRETO; CUNHA-FILHO,

38

2008).

1.2.3.2 Complexos de inclusão com ciclodextrinas

Os complexos de inclusão são compostos moleculares com a estrutura

característica de um aduto, em que um composto (designado como hospedeiro)

encerra outro no seu interior (o hóspede). A complexação ocorre quando uma

molécula hóspede preenche totalmente ou parcialmente a cavidade interna da

ciclodextrina que, devido ao caráter apolar, favorece a formação de complexos de

inclusão com moléculas hidrofóbicas. Ligações covalentes não são formadas ou

rompidas durante a formação do complexo de inclusão e, em soluções aquosas, os

complexos são prontamente dissociados (OLIVEIRA; SANTOS; COELHO, 2009).

Em solução aquosa, moléculas livres do fármaco estão em equilíbrio com

moléculas ligadas a ciclodextrinas. As características mais importantes dos

complexos são sua estequiometria e os valores numéricos de suas constantes de

estabilidade (K). Se m moléculas do fármaco (D, “drug”) se associam a n moléculas

de ciclodextrina (CD) para formar um complexo (Dm / CDn), então o seguinte

equilíbrio é alcançado (LOFTSSON; MÁSSON; BREWSTER, 2004):

(1)

O tipo de complexo de inclusão mais comum é o que possui estequiometria

1:1 (fármaco: ciclodextrina), no qual uma molécula do fármaco (D, do inglês drug)

forma um complexo com uma molécula de ciclodextrina (CD) (LOFTSSON;

HREINSDÓTTIR; MÁSSON, 2005):

(2)

Ressalta-se também que, para fármacos ionizáveis, a constante de

estabilidade é muito maior para a forma não ionizada em relação à forma ionizada

Portanto, pode-se melhorar a solubilização de fármacos ionizáveis em ciclodextrinas

através da modificação do pH (LOFTSSON; HREINSDÓTTIR; MÁSSON, 2007;

LOFTSSON; BREWSTER, 2012). A complexação também pode ser melhorada

através da formação de complexos ternários entre a molécula do fármaco,

39

ciclodextrina e um terceiro componente. A adição de pequenas quantidades de

polímeros solúveis em água ao meio de complexação, seguido de aquecimento em

autoclave, pode aumentar significativamente a constante de estabilidade do

complexo fármaco-ciclodextrina (MIRANDA et. al., 2011).

O resultado final é uma alteração das propriedades físico-químicas da

molécula-hóspede, incluindo sua solubilidade, estabilidade e biodisponibilidade. A

molécula da ciclodextrina pode proteger o fármaco do ataque de várias moléculas

reativas, reduzindo assim a hidrólise, oxidação, rearranjo estérico, racemização e

degradação enzimática dos fármacos (POPIELEC; LOFTSSON, 2017).

Existem vários métodos para o preparo de complexos de inclusão, como a

liofilização, pasta, mistura física, co-precipitação, atomização e fluidização

supercrítica. Dentre esses métodos, destaca-se a atomização, devido à maior

complexação das moléculas e menor tempo de preparo (DA CUNHA FILHO; SÁ-

BARRETO, 2007; IACOVINO et.al, 2017).

1.2.4 Caracterização do complexo de inclusão

Para avaliar a formação de complexos de inclusão com ciclodextrinas são

utilizadas técnicas físico-químicas que, em conjunto, provam a existência dessa

nova entidade: o complexo de inclusão fármaco-ciclodextrina.

1.2.4.1 Diagrama de solubilidade de fases

O diagrama de solubilidade de fases, desenvolvido por Higuchi e Connors

(1965), baseia-se na medição do efeito da complexação na solubilidade do substrato

e permite fazer inferências sobre a estequiometria de inclusão e estimar uma

constante relacionada com o grau de estabilidade do complexo formado. O método

“shake-flask” é amplamente utilizado para determinação da solubilidade

termodinâmica. Para a preparação da amostra, um excesso de fármaco é

adicionado ao meio de solubilidade, suficiente para produzir uma solução saturada

em equilíbrio com a fase sólida. O tempo para que o equilíbrio (entre o fármaco em

solução e o excesso de sólido) seja atingido depende da taxa de dissolução e o tipo

de agitação utilizada, por isso recomenda-se que um perfil de dissolução seja

realizado. Para a separação das fases das soluções saturadas, os dois métodos

40

mais utilizados são a filtração e a centrifugação. Em seguida, a concentração de

fármaco é determinada por método analítico adequado. Os valores obtidos

correspondem a solubilidade intrínseca do substrato (So) mais a quantidade do

fármaco dissolvida no complexo de inclusão, que é a solubilidade aparente

(JOUYBAN, 2010, p. 3).

O diagrama de solubilidade de fases é um gráfico (Figura 4) onde é

representada a solubilidade aparente do substrato em função da concentração da

molécula hospedeira, ou seja, é plotada uma curva da solubilidade do fármaco (eixo

y) versus a concentração de ciclodextrina (eixo x) (LOFTSSON; MÁSSON;

BREWSTER, 2004).

Figura 4 – Diagrama de solubilidade de fases.

Fonte: (DA CUNHA FILHO; SÁ-BARRETO, 2007).

Nos perfis classificados como tipo A, a solubilidade aparente do fármaco

aumenta em função da concentração de CD e três perfis são possíveis: AL, AP e AN.

No perfil AL, há um aumento linear da solubilidade com o aumento na concentração

de CD, ou seja, o complexo é de primeira ordem em relação a CD. O perfil AP

corresponde a um desvio positivo da linearidade, sendo o complexo de primeira

ordem em relação ao fármaco, mas de segunda ordem em relação a CD e, assim, a

ciclodextrina seria mais efetiva em concentrações elevadas. O perfil do tipo AN

corresponde a um desvio negativo, e sua interpretação é mais complexa devido a

interações entre soluto-soluto e soluto-solvente que podem ocorrer. Já nos perfis do

41

tipo B, há a formação de complexos com solubilidade aquosa limitada. No diagrama

do tipo BS, há inicialmente a formação de um complexo solúvel, com aumento da

solubilidade do substrato. Entretanto, a seguir, a solubilidade máxima é atingida e a

adição de mais CD forma um platô e, quando todo o substrato foi consumido, a

adição de CD forma um complexo insolúvel que precipita. No perfil do tipo BI, o

complexo é tão insolúvel que não há aumento na solubilidade aparente do substrato

(BREWSTER; LOFTSSON, 2007; DA CUNHA FILHO; SÁ-BARRETO, 2007).

No caso de perfil do tipo AL e assumindo-se que a estequiometria é do tipo

1:1, o diagrama também permite a obtenção da constante de estabilidade (Kc),

calculada a partir da inclinação da isoterma e da concentração intrínseca do

substrato (So), dada pela equação 3 (LOFTSSON; HREINSDÓTTIR; MÁSSON,

2005; BREWSTER; LOFTSSON, 2007; LYRA et al., 2010):

1 1 inclinação

So (1 - inclinação) ( )

Um método mais preciso para avaliar os efeitos das ciclodextrinas na

solubilização de fármacos, é determinar sua eficiência de complexação (EC). Para

complexos com estequiometria 1:1, a eficiência de complexação pode ser calculada

a partir da inclinação do diagrama de solubilidade de fases (Equação 4)

(LOFTSSON; HREINSDÓTTIR; MÁSSON, 2007):

EC So 1 1 D CD

CD

inclinação

1 - inclinação (4)

O valor de EC pode ser utilizado para calcular a razão molar fármaco:

ciclodextrina (D:CD), que pode ser correlacionada com o aumento esperado na

quantidade de formulação:

D CD 1 1 1

EC ( )

A equação 6 mostra a correlação entre o aumento no volume de formulação e

as massas moleculares (MW, do inglês molecular weight) da ciclodextrina (MWCD) e

do fármaco (MWDrug), e o valor de EC (LOFTSSON; HREINSDÓTTIR; MÁSSON,

2007):

42

Aumento no volume de formulação M CD

M Drug 1

1

EC (6)

As massas moleculares da CDs estão descritas na Tabela 1. Para encontrar o

novo “volume” de formulação, multiplica-se o resultado encontrado na equação 6

pela dose do fármaco.

1.2.4.2 Análise térmica

Os estudos de análise térmica, utilizando as técnicas de calorimetria

exploratória diferencial (DSC, do inglês Differential Scanning Calorimetry) e

termogravimetria (TG), permitem identificar mudanças na estabilidade térmica do

fármaco, podendo ser um indicativo da formação de um complexo de inclusão

(GIODARNO; NOVAK; MOYANO, 2001).

A técnica de DSC é a ferramenta analítica mais utilizada para avaliar as

interações entre fármaco e ciclodextrinas no estado sólido. A comparação entre as

curvas de DSC dos componentes individuais, sua mistura física e os complexos de

inclusão pode fornecer indicações em relação a modificações no estado sólido e

interações entre os componentes como consequência dos métodos usados na

preparação dos complexos e comprovar a real formação dos mesmos (MURA,

2015).

1.2.4.3 Difração de raios-X de pó (DRXP)

O emprego da técnica de difração por raios-X baseia-se na comparação dos

difratogramas das substâncias puras e do complexo formado. A difração de raios-X

de pó é a técnica cristalográfica mais empregada, devido a sua simplicidade e é

considerada uma das melhores técnicas para a caracterização de complexos de

inclusão. O perfil difratométrico dos complexos é comparado com o perfil dos

compostos separados e da mistura física, e eventos, como surgimento ou

desaparecimento de picos e mudança nas intensidades relativas, sugerem a

formação dos complexos (DA CUNHA FILHO; SÁ-BARRETO, 2007; TAKAHASHI et

al., 2012).

43

1.2.4.4 Espectroscopia no infravermelho com Transformada de Fourier

A espectroscopia no infravermelho com Transformada de Fourier (FT-IR) é

uma técnica bastante utilizada para avaliar a ocorrência de interações entre

diferentes moléculas que apresentam alteração do momento dipolo intrínseco,

provocado pela absorção da energia radiante, como consequência do seu

movimento vibracional ou rotacional. A caracterização dos complexos de inclusão é

baseada nos deslocamentos que ocorrem nas bandas de absorção da ciclodextrina

ou do fármaco, podendo ocorrer mudanças de posição, diminuição e mesmo

desaparecimento de picos característicos, causados pela ocorrência da

complexação (LYRA et al., 2010; TAKAHASHI et al., 2012; AGUIAR et al., 2014).

1.2.4.5 Dissolução

Os estudos de dissolução constituem um dos mais importantes testes in vitro,

uma vez que fornecem informações que permitem relacionar de forma mais estreita

a possível melhoria na biodisponibilidade do fármaco quando complexado com

ciclodextrinas. Este ensaio evidencia não somente os incrementos de solubilidade

intrínseca conseguidos pela complexação, mas também permite o estudo cinético da

liberação (SA BARRETO; CUNHA-FILHO, 2008).

1.2.4.6 CLAE

A cromatografia líquida de alta eficiência (CLAE) é um método amplamente

utilizado para se determinar com exatidão e precisão as constantes de associação

dos complexos de inclusão e sua estequiometria (MURA, 2014). Na literatura estão

disponíveis alguns métodos para a determinação de dronedarona (Tabela 2) em

materiais biológicos, matéria-prima e comprimidos. Dentre os métodos mais

utilizados para determinação de impurezas relacionadas e produtos de degradação

destacam-se a CLAE e cromatografia líquida acoplada a espectrometria de massas

(LC-MS). Nosso grupo de pesquisa desenvolveu um método por cromatografia

eletrocinética micelar (MEKC) para quantificação de dronedarona em comprimidos

(MARCOLINO et. al., 2013). Não existem métodos disponíveis na literatura para

determinação do fármaco em complexos de inclusão com ciclodextrinas até o

44

presente momento.

Tabela 2 – Métodos analíticos disponíveis na literatura para determinação de dronedarona

Referência Amostra Método analítico

Observações

Bolderman et. al., 2009

Plasma e miocárdio

CLAE -UV

Xie et. al., 2011 Plasma humano LC-MS/MS Fonte de ionização: APCI

Bin Jardan et al., 2014

Plasma de rato CLAE -UV Estudo farmacocinético

Ahirrao et. al., 2012

Comprimidos CLAE -UV

Tondepu et. al., 2012

Comprimidos e matéria-prima

CLAE -UV Impurezas relacionadas

Bhatt et. al., 2013

Comprimidos CLAE -UV

Landge et. al., 2013


CLAE -UV LC-MS

Impurezas relacionadas. Produtos de degradação nas condições ácida e

oxidativa. Fonte: ESI

Marcolino et. al., 2013

Comprimidos MEKC - UV

Pydimarry et. al., 2014


CLAE -UV e LC-MS

Fonte de ionização: ESI

Chadha et. al., 2016

Matéria-prima CLAE -UV e LC-MS/TOF

Identificação de produtos formados na hidrólise

alcalina e fotólise

1.2.5 Teste de citotoxicidade in vitro

Os estudos de segurança são uma etapa importante durante o

desenvolvimento de novas formulações. Desde a publicação do conceito dos R’s

(redução, refinamento e substituição) em 1959 por Russel & Burch em relação aos

experimentos com animais, foram desenvolvidos métodos alternativos à

experimentação animal, principalmente voltados a identificação de propriedades

tóxicas de produtos químicos (SPIELMANN, 2005). A avaliação de efeitos adversos

e desenvolvimento de toxicidade de novos produtos químicos pode ser realizada por

métodos in vitro, como uma etapa preliminar aos estudos de segurança toxicológica

in vivo, reduzindo assim a exposição dos animais aos produtos químicos e seu

45

sofrimento, e auxiliando os pesquisadores na tomada de decisão para modificar

estruturas químicas antes do seu reteste de toxicidade (MCKIM, 2010). Assim, os

ensaios de citotoxicidade in vitro têm sido empregados para elucidar questões

relativas aos mecanismos de toxicidade, pois muitos produtos químicos exercem seu

efeito tóxico sobre a estrutura e funções dos componentes celulares.

Os ensaios de citotoxicidade in vitro com linhagens celulares estabelecidas

são ferramentas úteis para o estudo dos efeitos toxicológicos de produtos químicos.

Dentre esses ensaios, o ensaio colorimétrico MTT é um dos métodos mais

comumente utilizados no estudo da viabilidade celular após a exposição a

substâncias tóxicas (NOGUEIRA et al., 2011). Esse teste de citotoxicidade é um

ensaio colorimétrico padrão para mensurar a proliferação celular, que também pode

ser utilizado para avaliar a citotoxicidade de fármacos e outros agentes tóxicos. O

sal MTT (brometo 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólico) é um reagente de

coloração amarela. O ensaio do MTT é baseado na redução desse sal, em células

viáveis, para produzir os cristais de formazan, um produto de coloração púrpura. O

MTT é reduzido na mitocôndria das células viáveis, através da clivagem da enzima

succinato desidrogenase, e a produção do formazan reflete o estado funcional da

cadeia respiratória. A quantidade de cristais formados é diretamente proporcional ao

número de células viáveis e, portanto, o teste pode ser utilizado para determinar a

viabilidade celular com precisão em estudos de citotoxicidade (MOSMANN, 1983;

BERRIDGE; TAN, 1993).

Os efeitos adversos fototóxicos de fármacos e cosméticos têm sido alvo de

preocupação de pacientes, dermatologistas e indústria farmacêutica. A fotorreação

geralmente ocorre em resposta à luz ultravioleta A (UVA) (315-400 nm) e abrange

dois fenômenos: fotoirritação (fototoxicidade) e fotoalergia. Reações fototóxicas

agudas tem sua intensidade máxima imediatamente após o início da reação e

decrescem até 48h e são semelhantes a queimaduras solares, com eritema,

infiltração e edema, podendo evoluir para queratose actínica e câncer de pele. Já a

fotoalergia é uma reação imunologicamente mediada e clinicamente é similar a

dermatite de contato, com eritema, infiltração e erupção cutânea (NEUMANN et al.,

2005). Ao nível celular, a radiação UVA causa estresse oxidativo, pois leva a

formação de espécies reativas de oxigênio (EROs), as quais exercem efeitos

nocivos incluindo oxidação de ácidos nucleicos, proteínas e lipídeos das membranas

(ZANATTA et. Al., 2010).

46

1.2.5.1 Ensaio de fototoxicidade

O teste de fototoxicidade in vitro 3T3 NRU (OECD TG 432) foi o primeiro

método validado aprovado pela OECD como alternativa ao uso de animais na

avaliação da fototoxicidade. Para avaliação da citotoxicidade, o ensaio utiliza cultura

de fibroblastos da linhagem 3T3 e a captação do corante vital vermelho

neutro,corante catiônico, que penetra na membrana celular por difusão passiva não

iônica e se acumula nos lisossomos. Após 24 h da aplicação do tratamento com a

substância química analisada e exposição à radiação, a citotoxicidade é

determinada pela redução da captação do corante vital, medida por

espectrofotometria. Esse ensaio baseia-se na comparação entre a citotoxicidade de

uma substância na presença e ausência de radiação simulando a luz solar.

Avalia-se a fototoxicidade através do cálculo do fator de fotoirritação (PIF, do

inglês Photo Irritation Factor), determinado pela relação entre a concentração do

produto que inibe a viabilidade celular em 50% (IC50) sem (Irr-) e com (Irr+)

exposição a doses não tóxicas de UVA (OECD, 2004):

As substâncias identificadas por esse teste são provavelmente fototóxicas in

vivo, após administração sistêmica e distribuição para a pele, ou após uso tópico. No

caso de não demonstrar potencial fototóxico no teste, as substâncias analisadas

estão desobrigadas a realizar futuros ensaios fototóxicos in vivo (OECD, 2004;

ZANATTA et al., 2010; LYNCH; WILCOX, 2011; KIM; PARK; LIM, 2015).

1.2.5.2 Determinação do potencial fotossensibilizante

A fotosensibilidade é semelhante a dermatite de contato, sendo ambas

reações de hipersensibilidade tardio tipo IV mediadas por células T específicas, que

se desenvolvem em duas fases definidas como fase de sensibilização e fase efetora.

O primeiro estágio do processo consiste na absorção dos fótons do comprimento de

onda apropriado pelas moléculas do fármaco, que atinge o estado excitado e

transfere a energia às moléculas de oxigênio, gerando as espécies reativas de

47

oxigênio. Após, o hapteno (fármaco fotoquimicamente modificado) se combina com

uma proteína carreadora, formado um antígeno completo. Em uma próxima etapa,

as células de Langerhans, células dendríticas imaturas residentes na pele,

internalizam o antígeno. As células de Langerhans ativadas migram para as áreas

corticais dos linfonodos regionais, onde se diferenciam em maduras e apresentam o

antígeno aos linfócitos T específicos, através das moléculas do complexo principal

de histocompatibilidade de classe II. Após estímulo apropriado, células T específicas

são produzidas com a capacidade de reagir ao antígeno, que na fase efetora,

migram para o sítio de inflamação, ativando-se e formando o eczema. Durante a

fase de sensibilização, as células de Langerhans se diferenciam e amadurecem,

expressando moléculas co-estimulatórias e de adesão, e secretando várias

citocinas, incluindo interleucina-1 e interleucina-8 (IL-8) (NEUMANN et. al., 2005;

ONOUE et. al., 2008; MITJANS et. al., 2010; MARTÍNEZ et. al., 2013). A IL-8 tem

um papel importante na fase efetora, não somente desencadeando o influxo de

leucócitos ao sítio da inflamação, mas também aproximando os leucócitos das

células dendríticas maduras (MITJANS et. al., 2008).

Considerando o mecanismo da reação de hipersensibilidade, foram

desenvolvidos ensaios (MITJANS et. al., 2008; MITJANS et. al., 2010) para a

identificação de alergenos utilizando a linhagem celular de leucemia monocítica

aguda humana (THP-1), visto que foi observado que essas células podem responder

especificamente aos sensibilizantes, através da expressão de moléculas co-

estimulatórias e produção de IL-8.

Martínez e col. (2013) desenvolveram um fotoensaio usando as células THP-1

e a liberação de IL-8 a fim de discriminar fotoirritantes e fotoalergenos. Após 24 h da

aplicação do tratamento com os fármacos e exposição a doses de UVA, a

viabilidade celular foi medida pelo ensaio do MTT. Clorpromazina foi utilizada como

controle positivo, pois é um fármaco fototóxico e fotoalergeno. O estudo propõe o

cálculo de índices de estimulação, calculados pela relação entre as liberações de IL-

8 a partir das células irradiadas (I-SI) e não-irradiadas (NI-SI) comparadas com os

controles não-tratados. Um índice de estimulação global, obtido pela relação entre

os dois índices (I-SI/NI-SI) foi proposto para discriminar os fármacos fotoalergenos

dos fármacos fotoirritantes.

48

1.2.5.3 Ensaios de citotoxicidade em células HepG2

Após a introdução da dronedarona no mercado, dois casos de lesões

hepáticas graves foram reportados, inclusive evoluindo para transplante de fígado.

Estudos conduzidos por Felser e col. (2013) investigaram o mecanismo associado a

hepatotoxicidade celular do fármaco utilizando mitocôndrias de fígado de rato, cultivo

primário de hepatócitos humanos e células tumorais de hepatoma humano (HepG2).

As investigações demonstraram que a dronedarona comprometeu a função

mitocondrial em concentrações entre 10 e 20 µM, e citotoxicidade foi observada a

partir de 20 µM. Os autores ressaltam que mesmo que as concentrações

plasmáticas do fármaco sejam baixas após administração oral (0,2 µM), o fármaco

sofre extenso metabolismo de primeira passagem, que conduz a uma baixa

biodisponibilidade (15%) e sugerem que as concentrações hepáticas possam ser

mais elevadas. O estudo concluiu que a dronedarona inibe a cadeia transportadora

de elétrons e a β-oxidação, bem como desacopla a fosforilação oxidativa nas

mitocôndrias hepáticas.

50

1.3 PROPOSIÇÃO

1.3.1 Objetivo geral

O presente trabalho teve por objetivo preparar e caracterizar complexos de

inclusão obtidos entre o fármaco dronedarona e as ciclodextrinas β-ciclodextrina (β-

CD) e 2-hidroxipropil-β-ciclodextrina (HP-β-CD), bem como realizar estudos de

segurança biológica por meio de ensaios de citotoxicidade in vitro.

1.3.2 Objetivos específicos

Preparar os complexos de inclusão dronedarona:ciclodextrinas utilizando

diferentes técnicas;

Validar método por cromatografia líquida de alta eficiência para determinação

do fármaco nos comprimidos e complexos de inclusão, e realizar estudo de

degradação forçada para avaliar a estabilidade desses em solução;

Caracterizar o fármaco, os excipientes e os complexos de inclusão

dronedarona:ciclodextrinas no estado sólido;

Avaliar o aumento de solubilidade do fármaco obtido através da formação dos

complexos de inclusão dronedarona β-CD e dronedarona:HP-β-CD;

Avaliar a citotoxicidade in vitro do fármaco e dos complexos de inclusão,

utilizando a linhagem celular 3T3 e o ensaio de viabilidade MTT;

Investigar o potencial fototóxico, fotossensibilizante e hepatotóxico utilizando

ensaios de citotoxicidade in vitro.

52

2. ARTIGO 1 - CINÉTICA DE DEGRADAÇÃO, ESTUDOS DE CITOTOXICIDADE

IN VITRO E VALIDAÇÃO DE MÉTODO POR CLAE INDICATIVO DE

ESTABILIDADE PARA CLORIDRATO DE DRONEDARONA EM COMPRIMIDOS E

EM COMPLEXOS DE INCLUSÃO COM CICLODEXTRINAS

Publicação científica: Marcolino, A.I.P; Scheeren, L.E; Nogueira-Librelotto, D.R.;

Fernandes, J.R.; Adams, A.I.H.; Carvalho, L.M.; Rolim; C.M.B. Degradation kinetics,

in vitro cytotoxicity studies and validation of a stability-indicating HPLC method for

dronedarone hydrochloride in tablets and in cyclodextrin inclusion complexes.

Manuscrito a ser submetido ao periódico Analytical Sciences (Fator de impacto:

1,174; Qualis: classificação B2).

54

INTRODUÇÃO

O presente capítulo tem como objetivo demonstrar os resultados do

desenvolvimento e validação de metodologia analítica para quantificação de

cloridrato de dronedarona em comprimidos comerciais e complexo de inclusão com

2-hidroxipropil-β-ciclodextrina. Foram realizados estudos de degradação forçada em

condições de estresse para verificar a formação de produtos de degradação, os

quais também foram analisados por cromatografia líquida acoplada à espectroscopia

de massas. Além disso, foram realizados estudos de citotoxicidade in vitro a fim de

investigar possíveis efeitos citotóxicos das amostras degradadas. Os ensaios

descritos neste capítulo foram realizados na Universidade Federal de Santa Maria,

no Laboratório de Pesquisa em Avaliação Biofarmacêutica e Controle de Qualidade

(LABCQ) e no Laboratório de Análises Químicas (LACHEM), com colaboração do

Prof. Dr. Leandro Machado de Carvalho.

56

o Original Paper

Degradation Kinetics, In Vitro Cytotoxicity Studies and Validation of

a Stability-Indicating HPLC Method for Dronedarone Hydrochloride

in Tablets and in Cyclodextrin Inclusion Complexes

Ana Isa P. MARCOLINO,* Laís E. SCHEEREN,* Daniele R. NOGUEIRA-LIBRELOTTO,*

Joana R. FERNANDES, ** Andréa I. H. ADAMS, * Leandro M. DE CARVALHO*** and

Clarice M. B. ROLIM*†

* Postgraduate Program in Pharmaceutical Sciences, Federal University of Santa Maria, Av.

Roraima n° 1000, Santa Maria – RS 97015-900, Brazil

** Department of Industrial Pharmacy, Federal University of Santa Maria, Av. Roraima n°

1000, Santa Maria – RS 97015-900, Brazil

*** Department of Chemistry, Federal University of Santa Maria, Av. Roraima n° 1000, Santa

Maria – RS 97015-900, Brazil

†Prof. Clarice Rolim.

E-mail: [email protected]

mailto:[email protected]

57

Abstract

The stability-indicating HPLC method for the determination of dronedarone

hydochloride in tablets and in inclusion complexes with cyclodextrin was carried out on a C18

column by using a buffer solution (0.3% glacial acetic acid; pH 4.9) and acetonitrile (35:65,

v/v) as mobile phase. Dronedarone was exposed to stress conditions, and drug degradation

kinetics was studied. The degraded samples were analyzed by mass spectrometry and the

preliminary toxicity against 3T3 cells was also determined. Inclusion complexation with

cyclodextrin reduced chemical degradation of dronedarone. The drug degradation kinetics in

alkaline conditions followed first-order reaction. Photodegraded samples presented cytotoxic

effects. Moreover, assay results were compared to a previously validated micellar

electrokinetic chromatography method, showing non-significant difference (p > 0.05). The

combination of HPLC, mass spectrometry and cytotoxicity study could be an important tool

for the screening of DRO pharmaceutical forms, thus improving quality and safety in the

development of novel drug delivery systems.

Keywords: Cyclodextrin, cytotoxicity, dronedarone, high-performance liquid

chromatography, inclusion complex.

58

Introduction

Dronedarone (DRO) is a non-iodinated benzofuran derivative structurally and

pharmacologically related to amiodarone, developed as an antiarrhythmic agent to overcome

the side effects of its parent compound. The molecular changes made to amiodarone to

produce DRO included the removal of iodine moiety and the addition of a methanesulfonyl

group, which conferred less lipophilic character (associated with reduced accumulation in

tissue) and less thyroid toxicity.1,2 In July 2009, the U.S. Food and Drug Administration (FDA)

approved DRO, in the film-coated tablet dosage form containing 400 mg, to reduce the risk of

cardiovascular hospitalization in patients with atrial fibrillation.3,4 Regarding its oral

administration, this biopharmaceutics classification system (BCS) class II drug (low solubility,

high permeability) exhibits food-effect and extensive first-pass metabolism, which lead to a

low absolute bioavailability (4-15%).5

Cyclodextrins (CD) are a group of cyclic oligosaccharides consisting of 6-8 α-D-

glucopyranose units linked by α-1,4 glycosidic bonds. These natural CD are named α-, β-

and γ-CD and present limited aqueous solubility. CDs derivatives, as hydroxypropyl-β-CD

(HP-β-CD), exhibit higher solubility and lower toxicity than the natural CDs, and are currently

used in many market products. CDs are shaped as a truncated cone with a central cavity,

due to the chair conformation of the glucopyranose units, which hydroxyl functions oriented

to the exterior confer a hydrophilic character to the outer surface, and the inner cavity lined

with skeletal carbons impart a lipophilic character. As a result, CDs have the ability to entrap

hydrophobic guest molecules inside their lipophilic central cavity, providing the formation of

inclusion complexes. In the pharmaceutical field, complexation with CDs is a promising

approach to enhance the aqueous solubility of poor-water soluble drugs and to improve their

bioavailability.6–8 Chemical stability of the drug could be enhanced by the formation of

inclusion complexes, as CD complexation has been shown to protect drug against hydrolytic,

oxidative and photo degradation.9

The need to investigate the potential of inclusion complexation phenomena stimulates

the development of new analytical methodology to assess the physicochemical properties

and stability of inclusion complex, which may be able to provide reliable information to

improve application and effectiveness of drug-CD inclusion complex.10

The literature survey revealed papers dealing with determination of DRO in biological

matrices by high-performance liquid chromatography coupled with ultraviolet detection11,12

and mass spectrometry.13 Some stability-indicating analytical methods have been reported

for the determination of DRO hydrochloride in bulk drug or pharmaceutical dosage form14,15

and related impurities,16–18 and to characterize the degradation products formed under

alkaline and photolytic conditions.19 Our research group developed a micellar electrokinetic

chromatography (MEKC) method to determine DRO hydrochloride in tablets.20 However, to

59

the best of our knowledge, the degradation kinetics of DRO was not reported in the literature

and there is no information regarding the cytotoxicity of the degradation products of DRO.

Furthermore, no analytical method to quantify DRO hydrochloride in inclusion complexes

with cyclodextrins has been published so far.

Considering that none of the most recognized pharmacopoeias include official

methods for the assay of DRO hydrochloride in the pharmaceutical dosage forms, the aim of

the present work was to develop a simple, reliable, accurate and stability-indicating HPLC

method for the quantitative analysis of DRO hydrochloride, according to the ICH guidelines,

with apparently the first degradation kinetic study. Additionally, a preliminary in vitro

cytotoxicity study of degraded DRO tablet samples was performed. Finally, the proposed

method was twofold; first, it has been successfully applied for the determination of DRO

hydrochloride in commercial film-coated tablets, establishing comparison with the validated

MEKC method; second, it allowed the possibility to assess DRO hydrochloride in cyclodextrin

inclusion complexes.

Experimental

Reagents and chemicals

DRO hydrochloride reference standard (assigned purity 99.7%) was purchased from

Sequoia Research Products (Pangbourne, Berkshire, UK). Film-coated tablets containing

400 mg of DRO hydrochloride (Multaq®, Sanofi, France) were obtained from commercial

sources. The tablets were labeled to contain the following excipients: hypromellose, starch,

crospovidone, poloxamer 407, lactose monohydrate, colloidal silicon dioxide, magnesium

stearate, polyethylene glycol 6000, titanium dioxide and carnauba wax, which were obtained

from different suppliers. HP-β-CD was obtained from Zibo Qianhui Biotechnology Co., Ltd.

(Zibo, Shandong, China). HPLC-grade acetonitrile and ammonium hydroxide were obtained

from Tedia® (Fairfield, OH, USA). LC-MS grade methanol, acetic acid, phosphate buffered

saline (PBS), dimethyl sulfoxide (DMSO), 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl)

tetrazolium bromide (MTT), and trypsin-EDTA solution (170,000 U L-1 trypsin and 0.2 g L-1

EDTA) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and

Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with L-glutamine (584 mg L-1)

and antibiotic/ antimicotic (50 mg mL-1 gentamicin sulphate and 2 mg L-1 amphotericin B),

were purchased from Vitrocell (Campinas, SP, Brazil). Ultrapure water was purified with

WaterPro™PS, Labconco system ( ansas City, MO, USA).

60

Apparatus and analytical conditions

HPLC method

The Shimadzu LC-20AT system (Kyoto, Japan) was equipped with a photodiode array

detector (PDA) (SPD-M20A) and auto sampler, and was operated with Shimadzu LC

Solution software (version 1.24SP1). A new aters XBridge™ C18 column (250 mm × 4.6

mm, 5 µm; Milford, MA, USA) maintained at room temperature (25 ± 1 °C), with a mobile

phase consisted of a buffer solution pH 4.9 (0.3% glacial acetic acid adjusted with

ammonium hydroxide):acetonitrile (35:65, v/v), at a flow rate of 1.0 mL min -1 was used. The

injection volume was 20 µL and PDA detector was set at 289 nm.

HPLC/APCI-MS method

The Agilent 6430 triple quadrupole mass detector, equipped with an atmospheric-

pressure chemical ionization (APCI) source was coupled with an Agilent 1260 Infinity LC-MS

chromatograph (Santa Clara, CA, USA) with automatic injection. Chromatographic

separation was achieved on a Poroshell® 120 EC-C18 column ( .0 100 mm; 2.7 μm; Santa

Clara, CA, USA) operated at room temperature, using a mobile phase of methanol:0.1%

glacial acetic acid (75:25, v/v), with a flow rate of 0.3 mL min-1. The main parameters were

optimized as follows: dry gas temperature, 325°C; vaporizer temperature, 200 °C; dry gas

flow, 5.0 L min-1; nebulizer pressure, 20 psi; capillary voltage, 2500 V; corona current

positive, 3 µA; charging electrode, 0 V; fragmentor voltage, 190 V, scan time, 500 ms.

Nitrogen was used as both the nebulizing and drying gases. The mass spectra were

recorded in positive ion mode. The data acquisition scanning range was from 100 to 600 m/z.

Preparation of reference solutions

The stock standard solution (500 µg ml-1) was prepared by dissolving 10 mg DRO

hydrochloride reference standard in 20 mL of methanol. This solution was daily diluted in

methanol up to adequate concentration and stored protected from the light at -20°C.

Preparation of sample solutions

Tablet solution. Twenty tablets containing 400 mg of DRO were accurately weighed,

combined and crushed to a fine powder. An amount of tablet powder equivalent to 20 mg of

DRO hydrochloride was transferred into 50 mL volumetric flasks, diluted in methanol and

sonicated for 15 minutes. An aliquot of the solution was filtered through quantitative filter

paper (Schleicher & Schuell) and diluted in methanol to obtain final concentration of 20 µg

61

ml-1. Samples were filtered through 0.45 µm membrane filter (Sartorius, Germany) before

analysis. The sample solutions were daily prepared.

Inclusion complex. The correspond amounts of DRO hydrochloride (molecular weight

593.2 g/mol) and HP-β-CD (molecular weight 1540.0 g/mol) equivalent to molar quantities in

the proportion 1:10 (1 M DRO: 10 M HP-β-CD) were mixed in a mortar for 10 min and then

dissolved in water at 50°C. After stirring for 20 min, the pH was adjusted for 4.5 with acetic

acid. Next, lactose was added as cryoprotectant (10%, p/v). The suspension was frozen for

24 h at -20°C and freeze-drying for 48 h. In order to determine the drug content, an aliquot of

the solid inclusion complex equivalent of 1 mg of DRO hydrochloride was diluted with

methanol and 400 µL of DMSO, sonicated for 15 min, and then diluted until the final

concentration of 20 µg ml-1.

Validation of the HPLC method

Validation was carried out assessing the following parameters: specificity, linearity,

precision, accuracy, limits of detection and quantitation and robustness according to the ICH

guidelines.21 The system suitability test was also performed to evaluate the reproducibility of

the chromatographic system, using six replicate injections of a reference solution. To

determine specificity, a placebo solution (an in-house mixture of all tablet excipients without

the active ingredient) was prepared.22

Forced degradation studies were performed to provide the stability-indicating property

and specificity of the method in accordance with the ICH guidelines.21,23 Stress testing was

performed by submitting a tablet solution (100 µg mL-1) under different stress conditions by

using the proportion 1:1 (v/v; tablet solution:degradant). Acid hydrolysis was performed in 3.0

M hydrochloric acid (HCl) at 80°C for 7 and 8 h using a water bath. Alkaline condition was

carried out with 1.0 M sodium hydroxide (NaOH) at 80°C for 0.5 and 1.0 h. The latter

solutions were cooled and neutralized with base or acid as needed. Photodegradation was

induced into transparent plastic cuvettes (Brand®; 12.5 mm × 45 mm × 12.5 mm) exposed to

near UV-A (spectral range of 352 nm; intensity approximately 1,350 W h/m²) light for 24 h

and UV-C (spectral range of 254 nm) light for 1.5 and 3.0 h in photostability UV chambers

(100 × 25 × 25 cm), at room temperature. Dark control samples were prepared for

comparison purposes, as recommended by the ICH guideline Q1B.24 At the end of each

exposure time, samples were diluted with methanol to final concentration of 20 µg mL-1 and

analyzed along with non-stressed sample by the HPLC method. The peak purity test was

carried out by PDA. The degraded tablet samples were also analyzed by the HPLC/APCI-MS

method. Inclusion complex and reference solutions (100 µg mL-1) were also submitted to the

stress testing under acidic (3 M HCl at 80°C for 7 h), alkaline (1 M NaOH at 80°C for 0.5 h)

62

and photolytic (UV-C light for 1.5 h) conditions and diluted until 20 µg mL-1 before analysis by

the HPLC method.

DRO degradation kinetics

The drug degradation under alkaline condition was determined by diluting the DRO

tablet, reference and inclusion complex solutions (100 µg mL-1) with 1.0 M NaOH, heated at

60°C in a water bath. After pre-established time intervals, samples were neutralized with 1.0

M HCl and diluted with methanol to final concentration of 20 µg mL-1. The DRO degradation

kinetics was monitored by the HPLC method.

In vitro cytotoxicity assay of degraded tablet samples

The murine Swiss albino 3T3 fibroblast cell line was grown in DMEM medium

supplemented with 10% (v/v) FBS, L-glutamine (584 mg L-1) and antibiotic/antimicotic (50 mg

mL-1 gentamicin sulfate and 2 mg L-1 amphotericin B), at 37 °C, 5% CO2. The 3T3 cells were

routinely cultured in 75 cm2 culture flasks and were harvested using trypsin-EDTA when the

cells reached approximately 80% confluence.

The cytotoxic effects of DRO degraded samples were measured following the

procedures previously described,25,26 using the MTT assay as the viability endpoint.27 DRO

tablet samples were submitted to accelerated degradation (hydrolytic and photolytic

conditions) as described in section “forced degradation studies”.

3T3 cells were seeded into the central 60 wells of a 96-well plate at a density of 1 x 105

cells/ml. After incubation for 24 h under 5% CO2 at 37 ºC, the spent medium was replaced

with 100 µl of fresh medium supplemented with 5% FBS containing the non-degraded and

degraded samples of DRO at the required concentration range (0.1 to 2. μg mL-1). As

control samples, medium containing methanol (0.1, 0.5, 1.0 and 2.5%) was also evaluated.

After 24 h, the sample-containing medium was removed, and 100 µl of MTT in PBS (5 mg

mL-1) diluted 1:10 in medium without FBS was then added to the cells. The plates were

further incubated for 3 h, after which the medium was removed, and 100 µl of DMSO was

added to each well to dissolve the purple formazan product. After 10 min shaking at room

temperature, the absorbance of the resulting solutions was measured at 550 nm using a

SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA) microplate reader. The effect of

each treatment was calculated as a percentage of cell viability inhibition against the

untreated control cells (cells incubated with medium only). Each cytotoxicity experiment was

performed at least three times in triplicate for each concentration tested. Results are

expressed as mean ± standard error of the mean (SE). Statistical analyses were performed

using one-way analysis of variance (ANOVA) to determine the difference between the sets of

63

data, followed by Dunnett’s posthoc test for multiple comparisons, using the SPSS® software

(SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant, and p <

0.005 were considered highly statistically significant.

Analysis of DRO in pharmaceutical formulation – Comparison between HPLC and MEKC

methods

The commercial pharmaceutical dosage form was analyzed by the HPLC method, as

previously described in the section “preparation of sample solutions”, and by a validated

MEKC method.20 Briefly, the experiments were carried out in a fused-silica capillary (50 µm

i.d.; 40.0 cm effective length), thermostatized at 30°C and using PDA set at 216 nm. The

running buffer solution consisted of 40 mM borate buffer and 50 mM SDS at pH 9.2. The

applied voltage was 28 kV. The samples containing 50 µg mL-1 of DRO reference and tablet

sample solutions were injected hydrodynamically at 50 mbar for 7s. The assay values

obtained by both methods were compared using statistical analysis by two-sample t-test.

Results

HPLC method validation

The specificity of the method was evaluated by the analysis of placebo and a solution

containing only HP-β-CD, both prepared as described in section “preparation of sample

solution”. Solutions containing the degradation products were also analyzed, which were

obtained in the forced degradation studies, performed in order to provide the stability-

indicating capability of the HPLC method. The chromatograms with the DRO degradation

behavior of the tablet, reference and inclusion complex solutions are shown in Figure 1.

Table 1 shows the % drug degradation for each stress condition.

64

Fig. 1 Chromatograms of DRO tablet solution (A), reference solution (B) and DRO inclusion

complex with HP-β-CD prepared by lyophilization (C) at 20 μg mL -1 showing peak 1 = DRO;

peaks 2,3,4 = degraded forms. (a) Non-degraded samples and samples submitted to stress

degradation conditions such as: (b) alkaline hydrolysis with 1 M NaOH at 80°C for 0.5 h; (c)

acidic hydrolysis with 3 M HCl at 80°C for 7 h and (d) after exposure to UVC light for 1.5 h.

(D) UV reference solution spectrum.

1.0 2.0 3.0 4.0 5.0 6.0

0

10

20

30

40

50

60

70

80

90

100mAU

min

1

1

1

1

(a)

(d)

(b)

(c)2

32

1.0 2.0 3.0 4.0 5.0 6.0 min

0

10

20

30

40

50

60

70

80

90

100mAU

(a)

(b)

(d)

(c)

1

1

1

1

2

4

3

B

C

4

1.0 2.0 3.0 4.0 5.0 6.0 min

0

10

20

30

40

50

60

70

80

90

100mAU

(d)

(a)

(b)

(c)

11

1

1

2 3

A

5

200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

0

25

50

75

100

125

150

175

200

225

250

mAU

217

288

D

65

Table 1 Amount of DRO degraded in each stress condition for tablet, reference and

inclusion complex solutions

Condition Tablet solution Reference

solution

Inclusion complex

solution

1.0 M NaOH at 80°C for 0.5

h

60% 57% 38%

3.0 M HCl at 80°C for 7 h 21% 14% 4%

UV-C for 1.5 h 24% 22% 2.5%

Initial assessment of alkaline degradation of DRO tablet sample in 0.1 M NaOH for 2

h showed no degradation, and under exposure to 1.0 M NaOH for the same period, drug

degradation was 7%. A complete (100%) drug degradation was obtained in 1.0 M NaOH at

80°C for 3 h. After 0.5 h of basic hydrolysis in 1.0 M NaOH at 80°C, additional peaks around

2.8-3.2 min (Fig.1A (b); peak 2) were detected. After 1 h of exposure to the same condition,

the drug degradation was 78%. For the free drug, a broad peak with tR of about 3 min was

observed (reference solution, Fig. 1B (b), peak 2). On the other hand, in the inclusion

complex, the additional peaks presented a lower intensity in comparison to the degraded

tablet and the free drug under the same conditions, as observed in Fig. 1C (b).

Acid degradation studies were initially performed with the tablet solution in 0.1 M and

1.0 M HCl for 2 h and no alterations were observed. Indeed, by using 1.0 M HCl maintained

at 60°C for 6 h, the peak area decreased only 2%. Therefore, more drastic conditions were

tested, and the reaction in 3.0 M HCl at 80°C for 7 h resulted in an additional small peak at

4.1 min, well resolved from the DRO peak (Fig. 1A (c); peak 3). After 8 h by using the same

condition, drug degradation was 43%. In the reference solution, a degrade form (peak 3) was

detected at 4.8 min. In the chromatogram of the inclusion complex (Fig. 1C (c), two additional

peaks were detected: the first, at around 2.8 min (peak 2) and the second at 4.8 min (peak

3).

Photodegradation of DRO was firstly studied after exposure to UV-A light for 8 h,

showing non-significant effects. In contrast, after 24 h drug exposure, degradation was

around 26%. After exposure to UV-C for 1.5 h, an additional peak was detected at 3.8 min

(Fig. 1A (d), peak 4). Almost 43% drug degradation was found following exposure to UV-C

light for 3.0 h. For the free drug in the reference solution, the degraded form (peak 4) was

observed at 3.8 and another at 4.6 min (Fig. 1B (d), peak 5). For the inclusion complex (Fig.

1C (d)), no additional peak was detected.

Table 1 shows the amount of DRO degraded in each stress condition for the tablet,

reference and inclusion complex solutions. From the results, it can be evidenced that the %

drug degradation for the inclusion complex were lower in comparison to the tablet and

66

reference solutions, for all the stress conditions, suggesting that the complexation with CDs

had a direct impact in DRO degradation, protecting the drug and enhancing the chemical

stability.

PDA analysis revealed that no formulation excipients and/or impurities were co-

eluting with DRO peak, showing peak purity index values higher than 0.9998. Likewise, the

resolution factor between the DRO peak and the nearest resolving peak was > 2,

demonstrating the ability to measure DRO in the presence of interferences.

The three analytical curves for DRO were constructed in the range 5 - 100 µg mL-1 for

the evaluation of linearity. The HPLC method was linear (r = 0.9999; y = 44111.42x +

21073.45, where x is the concentration and y is the absolute peak area). The analytical data

were validated by means of ANOVA, showing significant linear regression (p < 0.05) and

non-significant linearity deviation (p > 0.05).

LOD and LOQ were determined by using the mean of the slope (S) and the standard

deviation of the intercept (σ) of three independent curves, determined by a linear regression

line as 4260.02. The theoretical LOD and LOQ values, calculated according to ICH

guidelines21 as LOD . σ S and LOQ 10 σ S, were 0. 2 and 0.96 µg mL-1, respectively.

Experimentally, LOD was determined at the signal-to-noise ratio (S/N) of 3:1 and it was

found to be 1.0 µg ml-1. On the other hand, LOQ was determined at S/N ratio of 10:1, with

precision lower than 2%, and was set to be 5.0 µg ml-1.

The precision was determined by repeatability and intermediate precision, expressed

as relative standard deviation (RSD). Repeatability (intra-day) was determined by calculating

the RSD of assay results (%) of six independent sample preparations at 20 µg mL-1. The

intermediate precision was assessed by analyzing six samples at three different days (inter-

day) and by a second analyst (between-analysts). The precision results showed all RSD

values lower than 5.0% (Table 2).

Table 2 Intra-day and inter-day precision data for the proposed HPLC method

Intra-day Inter-day Between-analysts

Sample Day

Assaya (%)

RSD (%)

Mean assayb

(%)

RSDb

(%) Analyst

Assaya

(%) RSDc

(%)

Tablet 1 101.76 0.63 101.42 0.61 A 101.54 0.55 2 100.96 0.69 B 101.38 3 101.54 0.14

Inclusion complex

1 102.93 1.72 102.16 1.76 A 102.42 1.59 2 101.83 1.59 B 101.24 3 101.29 2.20

a. Mean of six replicates. b. (n = 18) c. Relative standard deviation between Analyst A and B.

67

Accuracy was evaluated by the recovery of known amounts of reference solution,

added to a sample solution (5.0 µg ml-1) to obtain sample solutions with final concentrations

equivalent to 50, 100 and 150% of the nominal analytical concentration of 20.0 µg ml-1.

Concerning the accuracy evaluation, the mean recovery for the three concentration levels

was found to be 99.30% (RSD = 0.59) for the tablet solution and 99.77% (RSD = 1.38) for

the inclusion complex solution, being each individual recovery value (Table 3) within the

desired range (100 ± 2%).28

Table 3 Recovery studies for the HPLC method

Sample Nominal

concentration (µg mL-1)

Added (µg mL-1)

Recovered (µg mL-1)a

Recovery (%)a

RSD (%)

Tablet 10 5.0 4.97 99.41 0.73 20 15.0 14.83 98.90 0.56 30 25.0 24.90 99.58 0.40

Inclusion complex

10 5.0 4.96 99.16 1.76 20 15.0 14.98 99.93 1.50 30 25.0 25.05 100.21 1.22

aMean of three determinations for each concentration.

Results of robustness studies, obtained after analyzing the tablet solution under

varied experimental values of flow rate, acetonitrile ratio and pH of the buffer solution, are

shown in Table 4. No significant changes were observed in the number of plates (N) and

tailing factor (Tf); however, as expected, total retention time (tR) varied between 4.2 and 6.4

min. Variations in the chromatographic conditions did not result in significant effects on DRO

assay results. Robustness was also evaluated after analyzing the inclusion complex by using

a 2-level 24-1 (eight experiments) fractional factorial design performed by the selection of the

same factors at high and low levels. Statistical evaluation was performed by evaluating the

response (assay of DRO in inclusion complex, %) processed by Minitab 17 statistical

software (Minitab Inc., State College, PA, USA). The Pareto chart showed the estimated

effect in decreasing order of magnitude, where the length of each bar was proportional to the

absolute value of the standardized effect divided by its standard error. The vertical line is the

critical limit for a α of 0.0 and is consider to establish which effects are statistically

significant. The analysis of the Pareto chart (Fig. 2) showed that none of the factors had a

significant effect on the quantification of DRO in the inclusion complexes, as the bars not

overtakes the vertical line, confirming the robustness of the HPLC method.

The stability of sample solutions was tested after 24 h storage at room temperature;

and statistical analysis performed with t-test showed no significant difference between initial

68

and 24 h assay values (p > 0.05), suggesting suitability for overnight analysis.

Table 4 The robustness testing of the HPLC method for DRO in tablets

Experiment Acetonitrile (%)

pH buffer

Flow rate (mL min -1)

Assay (%)

Tfa Nb tR

c

1 60 4.9 1.0 100.76 1.17 8648 6.0 2d 65 4.9 1.0 101.49 1.21 9154 5.9 3 70 4.9 1.0 100.18 1.23 7670 4.3 4 65 4.4 1.0 101.18 1.24 8219 5.3 5 65 5.4 1.0 101.17 1.14 10552 6.4 6 65 4.9 0.8 101.27 1.21 9608 6.2 7 65 4.9 1.2 99.54 1.21 7534 4.2

a. Tailing factor. b. Number of plates. c. Total retention time. d. Optimal conditions.

Fig. 2 Pareto chart obtained for the robustness assay of DRO in inclusion complex.

The system suitability test was carried out according to USP 3929 by determining the

retention factor, N and Tf, and values were found to be 2.34, 9154 and 1.21, with RSD values

of 0.11%, 1.21% and 0.17%, respectively. The results for peak area showed RSD values of

0.4 %, within the acceptable values (RSD ˂ 2.0%) indicating that the chromatographic

system was adequate for the analysis intended.

69

Analysis of degradation products by HPLC/APCI-MS

The mass spectra of the drug and degraded tablet samples are shown in Fig. 3. DRO

reference solution displayed a parent ion at m/z 557 [M+H+], which yielded fragmented ions

at m/z 101, 142, 170 and 435 in MS/MS studies. In the analysis of the degraded tablet

samples, the impurities were identified based on the presence or absence of peaks in

relation to those characteristic MS/MS spectra of the non-degraded DRO tablet sample. For

hydrolytic conditions, the mass spectral data for both acid and basic conditions showed a

protonated molecular ion at m/z 469 which formed two daughter ions at m/z 167 and m/z 202

in the product ion spectra. In the full scan mass spectra obtained for DRO submitted to

photolytic condition, the major molecular ion was observed at m/z 321, which yielded a

fragmented ion at m/z 127.

70

x106

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

+ Scan (2.356-2.471 min, 15 Scans)

556.6

C o u nts vs. Mass-to-Charge (m/z)100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

x104

0

1

2

3

4

5

6

7

+ Scan (3.641-3.723 min, 11 Scans)

469.8

166.9

410.8202.9

C o u nts vs. Mass-to-Charge (m/z)

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

x104

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2+ Scan (1.407-1.587 min, 23 Scans)

321.8

307.9

518.7

572.6

588.8

504.6283.8170.0


x105

0

0.2

0.4

0.6

0.8

1

1.2

1.4

+ Scan (3.747-3.829 min, 11 Scans)

469.6

166.9 256.0


100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

a

b

c

d

DRO

Acidic hydrolysis

Alkaline hydrolysis

Photolysis

71

Fig. 3 The full scan MS spectra (a-d) and product ion spectra (e-h) of [M+H]+ of DRO

reference substance (a) and degraded tablet samples obtained under: (b) acidic hydrolysis;

(c) alkaline hydrolysis and (d) after exposure to UV-C light.

x104

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

+ Product Ion (2.299-2.389 min, 12 Scans) (557.29999 -> **)

101.1

142.0

557.7170.0

435.8


x104

0

0.25

0.5

0.75

1

+ Product Ion:1 (3.637-3.707 min, 5 Scans) (469.60001 -> **)

167.0

202.9


100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400410420430440450460470480490500

x102

0

0.5

1

1.5

+ Product Ion:3 (3.489-3.620 min, 6 Scans) (469.89999 -> **)

202.7

167.0


100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400410420430440450460470480490500

x102

0

0.25

0.5

0.75

1

+ Product Ion:1 (1.440 min) (321.89999 -> **)

127.6

321.6


100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

e

f

g

h

DRO

Acidic hydrolysis

Alkaline hydrolysis

Photolysis

72

Kinetics of DRO degradation under alkaline conditions

The kinetic order of alkaline degradation after the hydrolysis of DRO tablet solution

with 1 M NaOH at 60°C was determined by plotting DRO concentration (zero-order), natural

log (first-order), and reciprocal (second-order) of DRO concentration versus time. The

kinetics data showed in Figure 4 suggested an apparent first-order reaction. The degradation

rate constant value obtained by the mathematical model was 0.242 h-1. The t90 (time where

10% of initial drug concentration is degraded) and half-life (t ½) were 0.438 h and 2.86 h,

respectively.

Fig. 4 Plots of concentration (a) zero-order reaction, natural log of concentration (b) first-

order reaction, and reciprocal of concentration (c) second-order reaction, against time, after

the hydrolysis of DRO tablet solution with 1.0 M NaOH at 60°C.

y = -2,6865x + 18,929R² = 0,9826

0

5

10

15

20

25

0 1 2 3 4 5 6

Co

ncen

trati

on (µg

mL

-1)

Time (hours)

y = -0,2327x + 2,9915R² = 0,9992

0

0,5

1

1,5

2

2,5

3

3,5

0 1 2 3 4 5 6Ln

of

co

nc

en

tra

tio

n(µ

g m

L-1

)

Time (hours)

y = 0,022x + 0,0438R² = 0,9775

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

0 1 2 3 4 5 6

1/c

on

cen

trati

on

Time (hours)

a

c

b

73

Cytotoxicity of DRO degraded tablet samples

The cytotoxicity assay results (Fig.5) revealed that the degraded samples obtained

after acidic and alkaline hydrolysis did not reduce cell viability significantly at all tested

concentrations. In contrast, the degraded samples obtained by photolysis, after 1.5 h and 3 h

of UV light exposure, showed potential cytotoxic effects at 2.5 and 1.0 µg mL-1, since the

statistical analysis revealed a significant difference among cell viabilities for these samples,

the negative control cells and lower concentration solutions.

Fig. 5 Cytotoxicity of DRO tablet solution before and after degradation treatments (acidic,

basic and photolytic stress conditions) on 3T3 cells as a function of concentration, as

determined by MTT viability assay. Concentrations tested (from left to right) of 2. μg mL-1

(blank), 1.0 μg mL-1 (striped), 0.5 μg mL-1 (black) and 0.1 μg mL-1 (gray). The data represent

the mean of three independent experiments ± SE (error bars). Statistical analyses were

performed using ANOVA followed by Dunnett’s multiple comparison test. * Statistically

different (p < 0.05) and ** highly statistically different (p < 0.005) from non-degraded sample.

Tukey’s multiple comparison test were also performed in order to verify if there is any

difference on the cytotoxicity between the degradation times. However, no statistically

significant differences were observed.

0

10

20

30

40

50

60

70

80

90

100

110

Control MeOH

Non-degraded sample

Photolysis 1.5 h

Photolysis 3 h

Basic Hydrolysis

0.5 h

Basic Hydrolysis

1 h

Acidic Hydrolysis

7 h

Acidic Hydrolysis

8 h

Ce

ll v

iab

ilit

y (

%)

*

**

* *

74

Analysis of DRO in commercial pharmaceutical formulation – Comparison between HPLC

and MEKC methods

The validated HPLC method was applied to the assay of DRO in the tablet dosage

form, giving mean assay results of 101.36 % ± 0.7540 (mean ± standard deviation; n = 12),

in agreement with the percent of label claim (95.0% to 105.0%).30 These assay results were

compared to those obtained with the validated MEKC method (100.82 % ± 0.6554; mean ±

standard deviation; n = 12). There was no significant difference between assay values

obtained by both methods (t-value obtained of 1.867; the critical value for t at α 0.0 0 was

2.074).

Discussion

In drug therapy, safety and efficacy are the fundamental issues of importance. Safety

is determined by the toxicological properties of the drug and impurities in bulk drug and

pharmaceutical form. In order to emulate the stress that the drug may be submitted during

manufacture processes and storage, forced degradation studies could be applied to provide

information on drug stability under different conditions, monitoring impurities as degradation

products.31,32 In this context, here the forced DRO degradation behavior in pharmaceutical

form was examined by using the HPLC and HPLC/APCI-MS methods.

For HPLC method development, preliminary separations were tested using two

brands of reversed phase C18 columns (250 mm × 4.6 mm, 5 µm), Phenomenex Luna® and

aters XBridge™. The latter provided acceptable theoretical plates (N) and resolution, with

symmetrical peak shapes combined with shorter analysis time. Reproducible separations

with an acceptable peak shape were achieved with buffer solution pH

4.9/methanol/acetonitrile mixture in different combinations; however, poor resolution and

longer analysis time were obtained. Then, considering DRO lipophilicity (calculated LogP =

6.3613), acetonitrile was selected as the organic modifier. Its effect on mobile phase

composition (ranging from 60 to 80%) was investigated. A better resolution and improved Tf

were obtained with 65% acetonitrile. The effect of pH of the buffer solution (0.3% glacial

acetic acid adjusted with ammonium hydroxide) was studied over a range from 3.8 to 6.0,

considering DRO higher solubility in acidic environment 33. A better Tf was obtained at pH

4.9, however, higher pH values resulted in broad peaks and peak tailing. DRO tR, and N

showed no significant changes in the range of pH 3.8-4.9. PDA was set at 289 nm due to

interference from the mobile phase at the first maximum wavelength 217 nm. Reference and

samples solutions were prepared in methanol due to higher DRO solubility in the solvent. For

75

the assay of the inclusion complex, DMSO was used to solubilize the CD and destabilize the

inclusion complex.34

Therefore, good chromatographic separation of DRO and its degradation products

with relatively short run time (7.0 min) was reached by using aters XBridge™ C18 column

with buffer solution pH 4.9 (0.3% glacial acetic acid adjusted with ammonium

hydroxide):acetonitrile (35:65, v/v), using isocratic elution. The proposed HPLC method was

accurate, linear, precise, robust and specific, with ability to separate DRO from its

degradation products and without any interference of formulation excipients. Additionally, the

obtained assay results for the tablets seemed to be in good agreement with those obtained

by the MEKC method, with similar accuracy (99.30±0.59 and 99.9±1.09, mean recovery % ±

RSD, for the RP-LC-PAD and MEKC methods, respectively) as well as the advantage of high

sensitivity (LOD of 0.32 µg mL-1 vs. 0.88 µg mL-1 and LOQ of 0.96 µg ml-1 vs. 2.66 µg ml-1 for

HPLC and MEKC20 methods, respectively).

Free DRO was highly susceptible to degradation under alkaline hydrolysis and heat.

Then, degradation kinetics was performed to elucidate the speed of this process with the

tablet solution. The alkaline DRO degradation kinetics seemed to follow first-order reaction,

indicating that the degradation rate is determined by one component concentration.35 These

findings might be useful during the manufacturing process of DRO drug products, suggesting

concerns in wet granulation process (involved in these tablets), the choice of optimal pH

range for solutions and the interaction with excipients, as these critical factors could promote

hydrolysis of the drug, compromising its stability and therefore efficacy. On the other hand,

DRO alkaline degradation from the inclusion complex was around 1.5-fold lower than the free

drug in reference and tablet solutions submitted to the same conditions. Another study of

inclusion complexes with low solubility drug has shown than complexation with CD reduced

drug degradation in aqueous solution under alkaline conditions.9 In relation to the photo

degradation, inclusion complexation reduced around 9-fold degradation in comparison to the

free drug in the reference and tablet solutions after exposure to UV-C light for 1.5 h, and the

degradation product could not be detected. A study of the inclusion effect of HP-β-CD on the

photochemical stability of fungicide pyrimethanil showed a significant decrease in its

degradation, indicating the photoprotective effect of the CD.36 Thus, the stabilization of the

drug by CD suggested that it could enhance drug stability and perhaps shelf-life of

pharmaceutical formulations.

During HPLC/APCI-MS method development, different atmospheric pressure

ionization techniques were evaluated. As the analytes were detected with low sensitivity by

using electrospray ionization (ESI) in positive and negative mode, the APCI mode was used,

which is a good ionization method for low-to medium-polarity compounds in liquid

chromatography.37 Considering that DRO has secondary and tertiary nitrogen groups in its

76

structure, with pKa value 9.4,13 analysis was performed in positive ion mode. Acetic acid was

used to improve sensitivity by favoring ionization of the analyte. Since acetonitrile was

associated with signal suppression in LC/MS-APCI applications,38 methanol was the organic

modifier of choice. By using the chromatographic condition methanol:0.1% glacial acetic acid

(75:25, v/v), DRO tR was 2.5 min.

The DRO reference substance, tablet sample and degraded tablet samples were

analyzed by the HPLC/APCI-MS method to increase knowledge about the possible

degradation products. For DRO reference substance, the mass fragments found in the study

were similar with those reported13 by using the same ion source. The fragments at m/z 170

and at m/z 435 were also described in a LC-MS-TOF study using positive mode of ESI and

were attributed to dibutylaminopropyl cation and the loss of CH2SO2, respectively.19

However, the fragmentation patterns of the degradation products do not match the pattern of

DRO. Likewise, in the degraded samples, the fragments did not match with those previously

reported by ESI studies.18,19 Probably, this could be related to the APCI source, which was

not previously used to analyze DRO degradation products. Then, these unknown peaks

obtained under acid and alkaline hydrolysis and photolysis were attributed to new

degradation products.

Cytotoxicity of DRO and its degraded tablet samples was assessed in 3T3 cells in

order to determine the potential toxicity of the degraded structures compared to the intact

molecule.26 The concentration range analyzed was selected considering a previous study,

which determined alterations in mitochondrial functions above 10 µM of DRO (5.932 µg mL-

1).39 DRO photodegraded samples presented cytotoxic effects at concentrations above 1.0

µg mL-1. In line with our experimental data, a case of photosensitivity reaction was reported

in a patient treated with the DRO commercial tablets.40 These results support the relevance

of employing cytotoxicity assays as an important tool during the preliminary studies for the

screening of chemicals, to foresee possible side reactions following exposure to degraded

samples.

Conclusions

Therefore, the overall results demonstrated that the combination of the stability-

indicating HPLC method, the HPLC/APCI-MS method and the cytotoxicity study played an

important role in detecting DRO degradation products, which could have a critical impact on

the quality of drug product and lead to safety and toxicological concerns. The proposed

HPLC method provided a simple and fast drug determination and could be applied in the

quality control and stability studies of DRO hydrochloride in tablets and in inclusion

77

complexes with cyclodextrins, allowing the development of new drug dosage forms with

enhanced chemical stability.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Brazilian National Council for Scientific and Technological

Development (CNPq) [grant numbers 401069/2014-1 and 447548/2014-0]; FAPERGS

(Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul) [grant number 2293-

2551/14-0]; and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

References

1. S. H. Hohnloser, H. J. G. M. Crijns, M. van Eickels, C. Gaudin, R. L. Page, C. Torp-

Pedersen, and S. J. Connolly, N Engl J Med., 2009, 360, 668.

2. J-Y. Le Heuzey, G. M. De Ferrari, D. Radzik, M. Santini, J. Zhu, and J-M. Davy, J

Cardiovasc Electrophysiol., 2010, 21, 597.

3. S. J. Connolly, A. J Camm, J. L. Halperin, C. Joyner, M. Alings, J. Amerena, D. Atar,

A. Avezum, P. Blomström, M. Borggrefe, A. Budaj, S-A. Chen, C. K. Ching, P.

Commerford, A. Dans, J-M. Davy, E. Delacrétaz, G. Di Pasquale, R. Diaz, P. Dorian,

G. Flaker, S. Golitsyn, A. Gonzalez-Hermosillo, C. B. Granger, H. Heidbüchel, J.

Kautzner, J. S. Kim, F. Lanas, B. S. Lewis, J. L. Merino, C. Morillo, J. Murin, C.

Narasimhan, E. Paolasso, A. Parkhomenko, N. S. Peters, K-H. Sim, M. K. Stiles, S.

Tanomsup, L. Toivonen, J. Tomcsányi, C. Torp-Pedersen, H-F. Tse, P. Vardas, D.

Vinereanu, D. Xavier, J. Zhu, J-R. Zhu, L. Baret-Cormel, E. Weinling, C. Staiger, S.

Yusuf, S. Chrolavicius, R. Afzal, and S. H. Hohnloser, N Engl J Med. 2011, 365, 2268.

4. U.S. Food and Drug Administration. FDA labeling information: Revised Multaq label,

http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/022425s025lbl.pdf

5. S.D. Han, S. W. Jung, S. W. Jang, M. Son, B. M. Kim, and M. J. Kang, Biol Pharm

Bull., 2015, 38, 1026.

6. P. Mura, J Pharm Biomed Anal., 2015, 113, 226.

7. S. S. Jambhekar, and P. Breen, Drug Discov Today., 2016, 21, 356.

78

8. Z. Fülöp, P. Saokham, and T. Loftsson, Int J Pharm., 2014, 472, 282.

9. A. Popielec, and T. Loftsson, Int J Pharm, in press.

10. P. Mura,  J Pharm Biomed Anal., 2014, 101, 238.

11. R. W. Bolderman, J. J. R. Hermans, and J. G. Maessen, J Chromatogr B., 2009, 877,

1727.

12. Y. A. Bin Jardan, R. Q. Gabr, and D. R. Brocks, Biomed Chromatogr., 2014, 28, 1070.

13. C. Xie, S. Yang, D. Zhong, X. Dai, and X. Chen, J Chromatogr B., 2011, 879, 3071.

14. V. K. Ahirrao, C. S. Patil, S. R. Bembalkar, M. V. Katariya, V. S. Sonnekar, R. P.

Marathe, R. B. Nawale, and R. P. Pawar, J Chil Chem Soc.,2012, 57, 1272.

15. K. K. Bhatt, E. M. Patelia, and I. Amin, J Anal Bioanal Tech., 2013, 4, 1.

16. N. Tondepu, S. S. Sait, K. V. Surendranath, R. K. Kaja, and S. Kumar, Am J Anal

Chem. 2012, 3, 544.

17. S. B. Landge, S. A. Jadhav, K. P. Nimbalkara, A. C. Mali, and V. T. Mathad, Am J Anal

Chem., 2013, 4, 323.

18. S. P. R. Pydimarry, V. K. Cholleti, and R. R. Vangala, J Chromatogr Sci., 2014, 52,

666.

19. R. Chadha, A. Bali, and G. Bansal, J Pharm Biomed Anal., 2016, 118, 139.

20. A. I. P. Marcolino, M. S. Sangoi, M. Steppe, A. I. H. Adams, and C. M. B. Rolim, Anal

Methods, 2013, 5, 3106.

21. ICH Q2 (R1). Validation of analytical procedures: text and methodology. Int Conf

Harmon. 2005;

22. R. C. Rowe, P. J. Sheskey, and M. E. Quinn, "Handbook of Pharmaceutical

Excipients", Pharmaceutical Press; American Pharmacists Association, 6th ed., 2009,

London ; Chicago : Washington, DC, 888.

23. ICH Q1A (R2). Stability Testing of New Drug Substances and Products Q1A(R2). In:

International Conference on Harmonization. 2003.

24. ICH Q1B. Stability testing: photostability testing of new drug substances and products.

Int Conf Harmon. 1996;

25. D. R. Nogueira, M. Mitjans, M. R. Infante, and M. P. Vinardell, Int J Pharm., 2011, 420,

51.

26. C. S. Paim, D. R. Nogueira, M. Mitjans, D. Ramos Lopez, J. de Lapuente Perez, M.

Steppe M, E. S. Schapoval, and M. P. Vinardell, Photochem Photobiol Sci., 2013,12, 805.

27. T. Mosmann, J Immunol Methods., 1983, 65, 55.

28. G. A. Shabir, J Chromatogr A., 2003, 987, 57.

29. United States Pharmacopeia. The United States Pharmacopeia and The National

Formulary. USP 39-NF 34. Rockville; 2016.

30. Australian Government, Department of Health and Ageing TGA, Australian Public

79

Assessment Report for Dronedarone Hydrochloride,

https://www.tga.gov.au/sites/default/files/auspar-multaq.pdf

31. D. Jain, and P. K. Basniwal, J Pharm Biomed Anal., 2013, 86, 11.

32. Z. Y. Liu, H. H. Zhang, X. J. Chen, X. N. Zhou, L. Wan, and Z. L Sun, Int J Mass

Spectrom., 2011, 303,90.

33. S. D. Han, S. W. Jung, S. W. Jang, H. J. Jung, M. Son, B. M. Kim, and M. J. Kang,

Chem Pharm Bull., 2015, 63, 295.

34. C. Mendes, A. Buttchevitz, J. H. Kruger, J. M. Kratz, C. M. O. Simões, P. O. Benedet,

P. R Oliveira, M. A. S. Silva, Eur J Pharm Sci., 2016, 83, 71.

35. S. Yoshioka, and V. J. Stella, "Stability of Drugs and Dosage Forms", Kluwer

Academic Publishers, 2002, New York, Boston, Dordrecht, London, Moscow.

36. C. Fernandes, I. Encarnação, A. Gaspar, J. Garrido, F. Borges, and E. M. Garrido, Int

J Photoenergy., 2014, 2014, 1.

37. R. Rebane, A. Kruve, P. Liigand, J. Liigand, K. Herodes, and I. Leito, Anal Chem.,

2016, 88, 3435.

38. A. Fredenhagen, and J. Kühnöl, J Mass Spectrom., 2014, 49, 727.

39. A. Felser, K. Blum, P. W. Lindinger, J. Bouitbir, and S. Krähenbühl, Toxicol Sci., 2013,

131, 480.

40. S. Kuo, K. Menon, and R. V. Kundu, Cutis., 2014, 94, E10.

80

Graphical Index

82

3 ARTIGO 2 – PREPARAÇÃO, CARACTERIZAÇÃO E ESTUDO DE

CITOTOXICIDADE DE COMPLEXOS DE INCLUSÃO DE DRONEDARONA E

CICLODEXTRINAS

Publicação científica: Marcolino, A.I.P; Fernandes, J.R.; Nogueira-Librelotto, D.R.;

Bender, C.R.; Wust, K.M.; Frizzo, C.P.; Mitjans, M., Vinardell, M.P.; Rolim; C.M.B

Preparation, characterization and cytotoxicity study on inclusion complexes of

dronedarone and cyclodextrins. Manuscrito a ser submetido ao periódico Materials

Science and Engeneering C (Fator de impacto: 4,164; Qualis classificação A2).

84

INTRODUÇÃO

Neste capítulo são apresentados os resultados da preparação de complexos

de inclusão de cloridrato de dronedarona com β-ciclodextrina e 2-hidroxipropil-β-

ciclodextrina, caracterizados por calorimetria exploratória diferencial, difração de

raios-X de pó, espectroscopia no infravermelho com Transformada de Fourier e

microscopia eletrônica de varredura. Além disso, foram realizados estudos de

dissolução em diferentes valores de pH e a determinação da solubilidade aquosa

dos complexos de inclusão. Por fim, foi avaliada a citotoxicidade in vitro do fármaco

e dos complexos de inclusão utilizando-se a linhagem celular 3T3 e o ensaio de

viabilidade do MTT. Os ensaios descritos neste capítulo foram realizados na

Universidade Federal de Santa Maria, no Laboratório de Pesquisa em Avaliação

Biofarmacêutica e Controle de Qualidade (LABCQ) e no Laboratório de

Farmacotécnica. Os estudos de análise térmica foram desenvolvidos no Núcleo de

Análises e Pesquisas Orgânicas (NAPO), sob orientação da Prof. Dra. Clarissa

Piccinin Frizzo. A caracterização por difração de raios-X de pó foi realizada no

Laboratório de Magnetismo e Materiais Magnéticos (LMMM), com a colaboração do

Prof. Dr. Gustavo Luiz Callegari e por espectroscopia no infravermelho, no

Laboratório de Materiais Inorgânicos, com colaboração do Prof. Dr. Sailer Santos

dos Santos. As análises por microscopia eletrônica de varredura foram realizadas

nos Centros Científicos e Tecnológicos da Universidade de Barcelona (CCiTUB),

Barcelona, Espanha, com colaboração da Prof. Dra. María Pilar Vinardell Martínez-

Hidalgo.

86

Preparation, characterization and cytotoxicity study on inclusion complexes of

dronedarone hydrochloride and cyclodextrins

Ana Isa Pedroso Marcolinoa, Joana Rodrigues Fernandesb, Daniele Rubert

Nogueira-Librelottoa,b, Caroline Raquel Benderc, Keli Maiara Wustc, Clarissa Piccinin

Frizzoc, Montserrat Mitjansd, María Pilar Vinardelld and Clarice Madalena Bueno

Rolima,b*

aPostgraduate Program in Pharmaceutical Sciences, Federal University of Santa

Maria, Av. Roraima 1000, 97105-900, Santa Maria – RS, Brazil

bDepartment of Industrial Pharmacy, Federal University of Santa Maria, Av. Roraima

1000, 97105-900, Santa Maria – RS, Brazil

cDepartment of Chemistry, Federal University of Santa Maria, Av. Roraima 1000,

97105-900, Santa Maria – RS, Brazil

dDepartment of Biochemistry and Physiology, Faculty of Pharmacy and Food

Science, University of Barcelona, Joan XXIII 27-31, 08028, Barcelona – Spain

* Corresponding author. Department of Industrial Pharmacy, Federal University of

Santa Maria, Santa Maria – RS 97015-900, Brazil. Tel.: (+55) 55 3220 8645. Fax:

(+55) 55 3220 8248.

E-mail address: [email protected] (C.M.B. Rolim).


87

Abstract

Dronedarone is a new antiarrhythmic drug for the treatment of atrial fibrillation. This

study investigates the complexation of dronedarone hydrochloride with β-cyclodextrin

(β-CD) and 2-hydroxypropil-β-CD (HP-β-CD) using three different techniques. The

complexes in the solid state were characterized by DSC, PXRD, FT-IR and SEM,

demonstrating the formation of the inclusion complexes and exhibiting different

properties from the pure drug. Its aqueous solubility increased about 4.0-fold upon

complexation with β-CD and HP-β-CD. The dissolution rate of the drug was notably

improved in all tested physiological pH values from 1.2 to 6.8 in the presence of both

cyclodextrins. Furthermore, an in vitro cytotoxic assay revealed that the inclusion

complexes could reduce the cytotoxic effects of the drug on 3T3 cells. The overall

results suggest that the inclusion complexes with β-CD and HP-β-CD may be

potentially useful in the preparation of novel pharmaceutical formulations containing

dronedarone hydrochloride.

Keywords: Dronedarone; cyclodextrin; inclusion complex; characterization;

dissolution; cytotoxicity

88

1. Introduction

Atrial fibrillation is the cardiac arrhythmia most commonly found in elderly

patients [1]. and as chronic disorder with a high rate of recurrence, it requires

continuous antiarrhythmic drug treatment to maintain normal sinus rhythm [2].

Available antiarrhythmic drugs, as amiodarone, are associated with adverse effects

as such as serious ventricular proarrhythmia, pulmonary and hepatic toxicity and

thyroid disorders [3,4]. Dronedarone (DRO) is a new antiarrhythmic drug structurally

related to amiodarone, but with a better safety profile, regarding thyroid and

neurological effects, by reducing accumulation in tissue [3–5]. In 2009, DRO was

approved by the Food and Drug Administration (FDA) and is provided as 400 mg

film-coated tablets, indicated to reduce the risk of hospitalization for atrial fibrillation

[6,7]. DRO hydrochloride is a poorly water-soluble molecule, practically insoluble in

water (0.64 mg/mL) and in buffers such as gastric fluid pH 1.2 and intestinal fluid pH

6.8 (<0.01 mg/mL) [8]; higher solubility (1-2 mg/mL) is achieved in a weak acid

medium (pH 3-5) [9]. In order to improve its pH-dependent aqueous solubility, the

manufacturer of the commercial table dosage form (Multaq®, Sanofi, France) used a

solid dispersion system with a triblock copolymer of polyethylene-propylene glycol [9].

Moreover, DRO absorption is influenced by food ingestion, which increases 2-3 fold

when the drug is given with a meal [10,11]. Due to food effect, absolute oral

bioavailability under fasting conditions is approximately 4%, however, a high fat meal

ingested with the drug could increase this value to 15%.

Cyclodextrins (CD) are cyclic oligosaccharides with a ring structure of α(1→4)-

linked glucose units, which shape is similar to a truncated cone, with a hydrophilic

exterior face and a lipophilic central cavity [12,13]. They have been used as

important pharmaceutical excipients, due its ability to form inclusion complexes,

89

where a lipophilic guest molecule is incorporated in the lipophilic central cavity [14] by

non-covalent interactions including hydrophobic interactions, electronic effects, steric

factors, hydrogen bonding and van der Waals forces [15,16].

In the case of poor soluble drugs as guest molecules, the formation of the

complex could impact in its physicochemical properties, increasing solubility and

dissolution rate [16], and promoting stability of the drug against degradation [17],

hence improving drug bioavailability [18]. Cyclodextrins also has been related to

reduced cytotoxicity [19].

β-cyclodextrin (β-CD) is the most employed CD due to its low cost of

production; however, its low solubility promoted the development of a derivative,

such as 2-hydroxypropil-β-CD (HP-β-CD), by substitution of hydroxyls in the rings of

the molecule, leading to an improved solubility and toxicological profile [20].

In this study, in an effort to improve DRO solubility, we investigated the

inclusion complexation of dronedarone hydrochloride with β-CD and HP-β-CD using

different techniques. This study included characterization of the inclusion complexes

by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder

X-ray diffraction (PXRD), Fourier-transform infrared spectroscopy (FT-IR) and

scanning electron microscopy (SEM), dissolution studies and an in vitro cytotoxicity

assay to evaluate the effects of free and complexed dronedarone on 3T3 cells

viability.

2. Material and methods

2.1 Material

DRO hydrochloride (purity > 98. %), β-CD and HP-β-CD were obtained from

90

Zibo Qianhui Biotechnology Co., Ltd. (Zibo, Shandong, China). Methanol and

acetonitrile (HPLC grade) were purchased from Tedia (Fairfield, OH, USA).

Phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), 2,5-diphenyl-3,-(4,5-

dimethyl-2-thiazolyl) tetrazolium bromide (MTT), and trypsin-EDTA solution (170,000

U L-1 trypsin and 0.2 g L-1 EDTA) were obtained from Sigma-Aldrich (St. Louis, MO,

USA). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM),

supplemented with L-glutamine (584 mg/L) and antibiotic/ antimicotic (50 mg/mL

gentamicin sulphate and 2 mg/l amphotericin B), were purchased from Vitrocell

(Campinas, SP, Brazil). All other reagents were of analytical grade. For all analyses,

ultrapure water was purified with aterPro™PS, Labconco system ( ansas City,

MO, USA).

2.2 High performance liquid chromatography method

The stability-indicating high performance liquid chromatography method

(HPLC), previously validated in accordance with the official guidelines, was

performed on a Shimadzu LC-20AT system (Kyoto, Japan) with Shimadzu LC

Solution software (version 1.24SP1). Analytical separations were carried out on a

aters XBridge™ C18 column (250 mm × 4.6 mm i.d., μm; Milford, MA, USA),

maintained at room temperature (25 ± 1 °C). The mobile phase consisted of a pH 4.9

buffer solution (0.3% glacial acetic acid adjusted with ammonium hydroxide):

acetonitrile (35:65, v/v), at a flow rate of 1.0 mL min-1. The injection volume was 20

μL. Photodiode array detector was set at 289 nm. For DRO quantification, analytical

curve were constructed with the reference solution in the concentration range from

2.5 to 25 µg mL-1. The limits of detection and quantification were 0.32 and 0.96 µg

mL-1, respectively.

91

2.3. Phase solubility studies

Phase solubility studies were carried out in order to investigate the effects of

β-CD and HP-β-CD on the solubility of DRO according to the method proposed by

Higuchi and Connors [21]. Excess amounts of DRO (10 mg) were added to solutions

of the β-CD and HP-β-CD with increasing concentrations (0-10 mmol/L). The

samples were incubated at 25 and 37°C and shaken on a rotary flask shaker (model

NT 712, Novatecnica, Piracicaba, SP, Brazil) for 48 h, time required to achieve

equilibrium according to a preliminary study. After that, samples were centrifuged at

4000 rpm for 10 min and the supernatant was filtered through a cellulose membrane

of 0.45 µm. The filtrated (100 µL) was transferred to volumetric flask, diluted with

methanol and concentrations of DRO were determined by the HPLC method. All

experiments were performed in triplicate.

The apparent stability constant (KC) was calculated from the slope of the

phase-solubility diagram and S0, the solubility of the drug in absence of CD, using the

equation 1 [21]:

The complexation efficiency (CE) is a more precise method to evaluate the

effect of the cyclodextrins on drugs solubility. CE can be calculated from the slope of

the phase solubility diagram, for complexes which stoichiometry of drug

(D)/cyclodextrin (CD) is 1:1, according to equation 2 [22]:

Eq. 1

Eq. 2

92

2.4 Preparation of complexes in solid state

The inclusion complexes of DRO (molecular weight 9 .2 g mol) and β-CD

(molecular weight 1135.0 g/mol) and HP-β-CD (molecular weight 1540.0 g/mol) in the

solid state were prepared by different techniques: freeze-drying (lyophilization), co-

lyophilization, and kneading followed by spray-drying. All binary mixtures were

prepared in the molar ratio of DRO to CD of 1:10 (g/mol). Samples were kept into the

desiccator until further analysis.

2.4.1. Physical mixtures

Physical mixtures of DRO and cyclodextrins were prepared by blending the

two components in a mortar until a homogeneous mixture was obtained in order to

control the complexation of DRO and cyclodextrins.

2.4.2. Lyophilization

The correspondent amounts of drug and each cyclodextrin were mixed

together for 10 min using a mortar. The mixture was dissolved in water and kept at

50ºC with moderate stirring for 20 min and then stirred at ambient temperature for 24

h. Lactose (10% p/v) was added to the obtained suspension, which was frozen at -

20ºC for 24 h and then freeze-dried for 48 h. The complex prepared with β-CD was

referred as LB and with HP-β-CD was referred as LH in the text.

2.4.3. Co-lyophilization

The mixture was dissolved in ethanol: water (1:1, v/v), kept at 50ºC for 20 min,

and then shaken for 24 h, at room temperature. Then, the organic solvent was

93

removed in a rotary evaporator at 50 ± 5ºC with stirring speed of 60 rpm. The

resulting suspension was frozen at -20ºC for 24h with lactose (10% p/v) and

lyophilized for 48 h. The complex with β-CD was referred as RB and with HP-β-CD,

RH in the text.

2.4.4. Kneading and spray-drying

The physical mixtures of DRO and β-CD or HP-β-CD were triturated in a

mortar for 20 min. A small volume of ultrapure water (0.5 mL) was added to the

mixture, which was mixed again for 5 min. The paste was added to 25 mL of water at

50ºC and stirred for 20 min. After that, the suspension was placed in a spray dryer

model LM MSD 1.0 (Labmaq do Brasil, Ribeirão Preto, SP, Brazil) with the following

operation conditions: inlet temperature: 120ºC, air pressure: 3 bar, feed flow rate:

0.21 L h. The complexes with β-CD and with HP-β-CD were named as SB and SH,

respectively.

2.5 Drug content determination

In order to determine the drug content of the inclusion complexes obtained

through different preparation methods, an amount of each complex equivalent to 1

mg of DRO was diluted with methanol and 0.4 mL of DMSO was added. The samples

were sonicated for 15 min, diluted appropriately and filtered (0.45 µm) before

analysis by the HPLC method.

2.6. Characterization

The inclusion complexes of DRO with crystalline (β-CD) and amorphous (HP-

94

β-CD) cyclodextrins obtained by different techniques were characterized in solid

state, in order to provide a comprehension of the properties of these new entities.

Likewise, pure DRO, β-CD, HP-β-CD and physical mixtures were analyzed with

comparison purposes.

2.6.1. Fourier-transform infrared spectroscopy (FT-IR)

The FT-IR spectra were obtained by mixture of the samples with potassium

bromide, according to the disk technique on a Bruker Tensor 27 FT-IR spectrometer

(Bruker, Germany), within the range 4000-400 cm-1 at a spectral resolution of 4 cm-1

and with an accumulation of 32 scans.

2.6.2. Powder X-ray diffraction (PXRD)

PXRD patterns were obtained with a Bruker D8 Advance (Bruker, Germany)

diffractometer system equipped with Cu- α radiation (λ 1. 4 Å) using a voltage of

40 kV, a current of 40 mA and at room temperature. The samples were analyzed in a

diffraction angle (2θ) range of -60° with a scan step size of 0.02 degrees and scan

speed of 1 degree/ s.

2.6.3. Differential scanning calorimetry (DSC)

Samples masses were accurately weighed (5 ± 0.001 mg) on a Sartorius M

500 P and placed in hermetically sealed aluminum pan and then analyzed on a

MDSC Q2000 (T-zero™DSC Technology, TA Instruments Inc., DE, USA) differential

scanning calorimeter, under dynamic nitrogen atmosphere (50 mL min-1; dry high

purity 99.999%). The instrument was initially calibrated in standard MDSC mode

95

using the extrapolated onset temperatures of melting indium (156.60°C) at a heating

rate of 10 °C min−1, and the heat from the fusion of indium (28.71 J g−1). Heat

capacity calibration was done by running standard sapphire (α-Al2O3) measurement

at the experimental temperature. The samples were subjected to three cycles of

heating and cooling (25°C to 200°C, 200°C to -80ºC, -80°C to 25°C), heated in a rate

of 10ºC min-1. DRO was heated up to 260°C and pure CDs up to 300°C.

2.6.4. Thermogravimetric analysis (TGA)

TGA curves were obtained with a TGA Q5000 (TA Instruments Inc., USA),

under N2 flow (40 mL min-1). The temperature range was 40°C to 900°C, with a

heating rate of 10°C min-1.

2.6.5. Scanning electron microscopy (SEM)

A small amount of the samples was placed on a brass stub using conductive

adhesive tape. Later, the samples were coated with a thin layer of gold

(approximately 40 nm) using a JFC-1100 (JEOL, Tokyo, Japan) ion sputter coater in

order to improve their electrical conductivity. The observation was made using a

JSM-6510 (JEOL) scanning electron microscope with an acceleration voltage of 15

kV.

2.7. Determination of DRO aqueous solubility after complexation

In order to investigate the increase on DRO solubility after complexation,

excess amount of pure DRO (10 mg) and inclusion complexes (around 500 mg;

equivalent to 10 mg of DRO) were added to 3 mL of water. The suspensions were

stirred at 25°C for 24 h. Samples were filtered through 0.45 membrane filters and

96

analyzed for DRO content by the HPLC method.

2.8. Dissolution studies

The dissolution studies of DRO, β-CD and HP-β-CD and inclusion complexes

DRO β-CD and DRO: HP-β-CD were performed using the USP dissolution

apparatus, type II.

10 mg of free dronedarone or equivalent amount of inclusion complexes were

separately added to 900 mL of dissolution media at 37 ± 0.5 ºC in a PharmaTest®

multi-bath (n = 6) dissolution system (Hamburg, Germany), by using standard USP

apparatus II (paddle) with a stirring rate of 75 rpm. Simulated gastric fluid pH 1.2

(consisting in 2 g NaCl and 7 mL of HCl in 1000 mL of distilled water), acetate buffer

pH 4.5 and phosphate buffer pH 6.8 were used as dissolution media to investigate

the dissolution properties of the complexes. The samples were withdrawn (5 mL) at

pre-determined time intervals, and the same volume of fresh medium was replaced.

The samples were filtered, diluted with methanol at the final concentration of 10 µg

mL -1 and analyzed by the HPLC method.

2.9. Chemical stability

The inclusion complexes of dronedarone with β-CD and HP-β-CD freshly

prepared and analyzed on the first day (D0) were divided in two aliquots and stored in

a climate stability chamber (Mecalor, São Paulo, SP, Brazil) at 40°C and 75% relative

humidity and into the desiccator at room temperature. After 30 days of storage (D30),

the samples were analyzed by PXRD and the content was determined by the HPLC

method.

97

2.10. In vitro cytotoxicity assay

2.10.1. Cell culture

The murine Swiss albino 3T3 fibroblast cell line was grown in DMEM medium

supplemented with 10% (v/v) FBS, L-glutamine (584 mg L-1) and antibiotic/antimicotic

(50 mg mL -1 gentamicin sulfate and 2 mg L -1 amphotericin B), at 37 °C, 5% CO2.

The 3T3 cells were routinely cultured in 75 cm2 culture flasks and were trypsinized

using trypsin-EDTA when the cells reached approximately 80% confluence.

2.10.2. Sample preparation

An amount of inclusion complexes of dronedarone with β-CD and HP-β-CD

equivalent to 1 mg of DRO was diluted in purified water in order to obtain a stock

solution at 100 μg mL-1 DRO. For pure DRO, due to its low solubility in water, the

stock solution was prepared with methanol. An amount of 10 mg of DRO was diluted

with methanol in a 5 mL flask. From this solution, dilutions were made in water to

prepare a stock solution at 100 μg/mL, containing 20% of methanol. Aliquots of stock

solutions were diluted latter in cell culture medium to obtain final concentrations of

1.25, 2.50 and 5.00 μg mL -1.

2.10.3. Chemical exposure and MTT assay

The cytotoxic effects of free drug and the inclusion complexes of DRO with β-

CD and HP-β-CD were measured following the procedures previously described

[23,24], using the MTT assay as the viability endpoint [25]. 3T3 cells were seeded

into the central 60 wells of a 96-well plate at a density of 1 x 105 cells mL -1. After

incubation for 24 h under 5% CO2 at 37 ºC, the spent medium was replaced with 100

98

µL of fresh medium supplemented with 5% FBS containing free DRO and the

inclusion complexes at the required concentration range (1.25 to 5.00 μg mL -1). As

control samples, medium containing methanol (0.25, 0.5 and 1.0%) was also

evaluated. After 24 h, the sample-containing medium was removed, and 100 µL of

MTT in PBS (5 mg mL -1) diluted 1:10 in medium without FBS was then added to the

cells. The plates were further incubated for 3 h, after which the medium was

removed, and 100 µL of DMSO was then added to each well to dissolve the purple

formazan product. After 10 min shaking at room temperature, the absorbance of the

resulting solutions was measured at 550 nm using a SpectraMax M2 (Molecular

Devices, Sunnyvale, CA, USA) microplate reader. The effect of each treatment was

calculated as a percentage of cell viability inhibition against the untreated control

cells (cells incubated with medium only).

2.11. Statistical analysis

Each cytotoxicity experiment was performed at least three times using three

replicate samples for each concentration tested. Results are expressed as mean ±

standard error of the mean (SE). Statistical analyses were performed using one-way

analysis of variance (ANOVA) to determine the difference between the sets of data,

followed by Dunnett’s and Tukey’s posthoc test for multiple comparisons, as

indicated, using the SPSS® software (SPSS Inc., Chicago, IL, USA). p < 0.05 was

considered statistically significant, and p < 0.005 were considered highly statistically

significant.

99

3. Results and Discussion

3.1. Phase-solubility diagram

The phase solubility diagrams of DRO in aqueous solutions of β-CD and HP-β-

CD were the first technique used to verify if the increment of cyclodextrin

concentration increases the apparent solubility of DRO. The selection of the

quantitative method to construct the phase solubility diagram considered that

commonly used techniques such as UV-Vis spectrometry are not suitable for

mixtures [26]; hence, we chose a reversed-phase liquid chromatography method to

determine DRO aqueous solubility. The graphic representations were constructed by

plotting the total molar concentration of DRO found against the total molar

concentration of CD added. Through the analysis of isotherms obtained at 25°C (Fig.

1a), it was found that the solubility of DRO increased linearly as the concentration of

both cyclodextrins increased. The diagram obtained by the inclusion complex with

HP-β-CD seemed to be AL-type, as established by the Higuchi and Connors model

[21].

The intrinsic solubility (S0), maximum solubility (Smax), and solubility efficiency

(SE: Smax/ S0) and slope values are also presented in Table 1. From each diagram

(Fig. 1a), stability constant (KC) and complexation efficiency (CE) were calculated,

according to Eq. 1 and 2, respectively.

100

Fig. 1. Phase solubility diagrams of DRO with β-CD (blue) and HP-β-CD (red) in

aqueous solution at (a) 25 °C and (b) 37 °C.

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0 2 4 6 8 10

[Dro

ned

aro

ne]

(mM

)

[CDs] (mM)

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0 2 4 6 8 10

[Dro

ned

aro

ne]

(mM

)

[CDs] (mM)

β-CD HP-β-CD

a

b

101

Table 1 – Results of DRO intrinsic solubility (S0), maximum solubility (Smax), and

solubility efficiency (SE), slope and stability (KC) and complexation efficiency (CE)

constants, from phase solubility diagrams at 25°C.

Cyclodextrin S0

(mg/mL)

Smax

(mg/mL)

SE Slope KC (M-1) CE

β-CD 0.73 ±

0.03

2.35 ± 0.18 3.2 0.2722 303.8 0.3728

HP-β-CD 0.73 ±

0.03

2.38 ± 0.15 3.3 0.2867 327.4 0.4019

The analysis of solubility efficiencies (SE, Smax/ S0) of the complexes showed

an enhancement in the DRO solubility of three times, as a result of the interaction

with both CDs. For the calculation of the stability constant (Kc), the drug solubility in

absent of cyclodextrin was experimentally determined, as the drug intrinsic solubility

(S0) value. The KC values allow comparing the drug affinity with different

cyclodextrins and the most common values are between 50 and 2000 M-1 [27]. Lower

values suggest instable complexes, and very high values (> 10000 M-1) suggest that

the drug is strongly linked, which can reduce its bioavailability [28]. The results of

Table 1 shown that KC values are within the desired range, moreover, it was found

that DRO affinity was slightly higher with HP-β-CD.

The solubilization efficiency, referred as complexation efficiency (CE), is a

more accurate method, since it only depends on the slope of the diagram and is

independent of S0 values, and as a result, there was less variability of KC values [22].

In relation to the CE values obtained in this study, a slightly higher value was

obtained for HP-β-CD, indicating that this CD is a better solubilizer than β-CD. The

102

values of CE shown in Table 1 are similar with those previously reported in a study

which determined the CE values of 13 inclusion complexes with HP-β-CD and

different drugs, with results of 0.39 ± 0.47 (mean ± standard deviation) [22].

The phase solubility diagrams obtained at 37°C (Fig. 1b) suggested that the

inclusion complexes formed were soluble in water, with higher solubility than the non-

complexed drug. A slight negative deviation from linearity indicates the diagram AN

type. To this diagram could be attributed some theories including alterations

transmitted to the solvent from the solubilizing agent; dielectric constant changes in

complexation media induced by the CD, modifications of complex solubility and self-

association of CD molecules [29].

3.2 Determination of drug content in the solid state

In the formulation development, inclusion complexes of DRO with β-CD and

HP-β-CD were prepared at different molar ratios of drug to CD of 1:1, 1:2, 1:5 and

1:10 (g/mol) by the three different techniques. The product obtained with the molar

ratio of drug to CD of 1:10 (g/mol) was found to have the higher DRO content, and,

therefore, this was the chosen stoichiometry. The drug content in the inclusion

complexes of DRO with β-CD and HP-β-CD were determined as described in section

2.5, by using DMSO in order to solubilize the cyclodextrins and destabilize the

complex, followed by dilution with methanol to solubilize DRO. The drug content of

each inclusion complex was calculated considering the respective yield (%), by

comparison with a DRO reference solution (Table 2). All the preparation techniques

achieved drug contents higher than 85% in the freshly prepared (D0) complexes in

the molar ratio drug to CD of 1:10 (M/M). The slightly lower DRO content for the

inclusion complex with β-CD prepared by the colyophilization method (RB) could be

103

due to the partial drug precipitation during the evaporation process.

The percent yield obtained for each preparation technique was also presented

in Table 2, represented as the percent of the recovered powder obtained by each

technique calculated by the relation between the final and initial weight of the

powder. Lyophilization presented a higher yield in comparison to the spray-drying

technique, which usually is attributed a lower yield as a result of the loss of the very

fine powder that could not settle on the cyclone collector chamber [30]. The inclusion

complex with β-CD prepared by this technique revealed a tendency to adhere to the

cyclone wall, which could explain the lowest % yield.

Table 2 – Drug content of inclusion complexes of DRO with β-CD and HP-β-CD

obtained by the HPLC method and yield of each preparation technique.

Inclusion

complexa

Yield (%) Drug content

(%) at D0b

Assay (%) at D30

kept in stability

chamberb

Assay (%) at D30

kept in room

temperatureb

LB 94 ± 2 92.99 ± 1.6 86.21 ± 3.1 83.99 ± 3.1

LH 94 ± 3 91.58 ± 0.2 78.40 ± 2.0 88.35 ± 2.3

RB 92 ± 1 85.58 ± 0.4 85.21 ± 0.9 83.29 ± 0.6

RH 97 ± 1 91.99 ± 1.7 85.85 ± 2.8 90.91 ± 2.3

SB 37 ± 5 93.69 ± 1.4 84.33 ± 4.1 91.88 ± 0.8

SH 55 ± 7 95.93 ± 1.4 90.11 ± 2.6 92.28 ± 2.5

aInclusion complexes obtained by lyophilization with β-CD (LB) and HP-β-CD (LH), by colyophilization with β-CD (RB) and HP-β-CD (RH) and by kneading following spray-drying with β-CD (SB) and HP-β-CD (SH). bResults are presented as mean ± S.D. of two experiments.

104

3.3 Characterization

The characterization methods were applied to study the differences among the

three preparation techniques of the inclusion complexes with β-CD and HP-β-CD in

the solid state.

3.3.1 Differential scanning calorimetry (DSC)

The samples were submitted to DSC analysis to study their thermal behavior.

The DSC curves of pure DRO, β-CD, HP-β-CD, physical mixtures and inclusion

complexes obtained by the different techniques are shown in Fig. 2. The

observations from the curves were represented in Table 3. DRO showed a unique

and well defined endothermic peak at 144.40°C, relative to its melting point, which

suggests its crystalline state (Fig. 2a). β-CD exhibited two endothermic peaks: the

first at 118°C, related to loss of water from cyclodextrin cavity and the second at

223°C (Fig. 2b). There is no agreement concerning the nature of this last transition,

but some authors attributed this endothermic effect to a reversible transformation of

β-CD in the solid state [31]. The melting point of β-CD is referred as an endothermic

peak near 325°C [32,33]. In the DSC curve of HP-β-CD, the loss of water molecules

from the CD cavity was observed as a broad large peak near to 80°C (Fig. 2c);

dehydration has also been observed for amorphous cyclodextrins [34].

105

a

b

b

c

c

d

d

e

106

i

g

f

h

j

107

Fig. 2 – DSC curves obtained for DRO (a), β-CD (b), HP-β-CD (c), physical mixture

with β-CD (d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with

β-CD (f) and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by

kneading following spray-drying with HP-β-CD (j) and β-CD (k) (10 °C.min-1 variations

in temperature and in nitrogen atmosphere).

As shown in Fig. 2d and 2e, the physical mixtures of DRO with β-CD and HP-

β-CD showed melting points close to the pure drug. The inclusion complexes of DRO

with β-CD prepared by lyophilization and colyophilization (Fig. 2f and 2h,

respectively) presented Tfus higher than the free drug, with values between the drug

and the β-CD melting points, indicating a solid state modification and an interaction

between the components, as a consequence of the formation of a inclusion complex.

These complexes also presented Tg in the first cycle heat/cold. In the DSC curve of

the complex prepared by spray-drying (Fig. 2k), the melting peak of DRO

disappeared, providing evidence of inclusion complex formation [35], which was also

confirmed by the PXRD analysis.

In all the inclusion complexes of DRO with HP-β-CD, the melting points and

k

108

enthalpies of DRO were altered; the melting points were shifted toward higher

temperatures and lower ∆H°fus1 values were observed. The inclusion complexes

obtained through colyophilization (Fig. 2i) and spray-dryer techniques (Fig. 2j)

presented Tc, which suggests the formation of a new crystalline phase. Only the

inclusion complex obtained through spray-drying presented Tg in the first heat/cold

cycle. The alterations of the melting points of DRO found in the inclusion complexes

with HP-β-CD can be related to the conversion of DRO from crystalline to amorphous

form, as shown in PXRD analysis, which could be attributed to the complexation.

Afterwards, thermogravimetric analysis (TGA) was performed to confirm the

loss of water from CD cavities. TGA analyses were conducted with β-CD, HP-β-CD

and the pure drug to determine the loss of mass that occur in function of the increase

of temperature. As shown in Table 4, both cyclodextrins presented the first

temperature of decomposition between 30°C and 119°C. These values could be

related to the loss of water adsorbed on the cyclodextrins (4.61 and 9.70 wt.%, for

HP-β-CD and β-CD, respectively), which were evaporated during the initial phase of

heating. For β-CD, the second initial temperature of decomposition started around

250°C with a loss in mass of 78% from this temperature, as evidenced for natural CD

[35]. For HP-β-CD, decomposition takes place above 286°C with mass loss of 89%,

with a slightly higher thermal stability than parent CD [31].

109

Table 3 – Thermal analyses obtained by DSC for dronedarone, β-CD, HP-β-CD, physical mixtures and inclusion complexes prepared by different techniques.a

Samples Tfus

b (°C)

∆H°fusc

(kJ g-1) Tc

d

(°C) ∆H°c

e

(°C) Tg

f

(°C)

DROg 144 ±1

81.5 ±11.7

-

Physical mixture with β-CD 148 65.9

Physical mixture with HP-β-CD 151 57.1

LBg 193 80.1 159

LH 189 67.5

RB 194 111.9 139

RH 188 73.4 131 33.9 -

SH 172 36.3 143 41.6 52

SB 89 146.0

a Thermal events observed in the first heat/cool cycle. b Melting temperature in the first cycle. c Melting enthalpy in the first cycle. d Crystallization temperature. e Crystallization enthalpy. f Glass transition temperature. gMean ± SD of three experiments. For β-CD, a first endothermic event at 122 ± 6°C (∆H°fus = 343±60 kJ g-

1) and a second endothermic event at 223 ±1°C (∆H°fus = 7.34±3.28 kJ g-1) were observed. For HP-β-CD, an endothermic effect was observed at 80±12°C (∆H°fus =

128±29 kJ g-1).

Table 4 - Thermal analysis by TGA to β-CD, HP-β-CD and DRO.a

Compound Ti1

b (°C)

Tf1c

(°C) Td1

d (°C)

%1e

Ti2 b

(°C) Tf2

c (°C)

Td2d

(°C) %2

e

β-CD 32.0 119.5 78.9 9.7 248.1 623.4 325.6 78.2

HP-β-CD 30.8 98.7 40.1 4.6 286.8 488.2 362.5 89.4

DRO - - 288.7 - 166.8 595.0 329.4 87.4

a Heating rate of 10 °C min-1. b Initial decomposition temperature. c Final decomposition temperature. d Decomposition temperature. e Percentage weight loss (wt.%).

110

3.2. Powder X-ray diffraction (PXRD)

The PXRD patterns of DRO, β-CD, HP-β-CD, physical mixtures and inclusion

complexes with crystalline (β-CD) and amorphous (HP-β-CD) CD obtained through

the different preparation methods (lyophilization, colyophilization and spray-drying)

are presented in Fig. 3, with the respective angles (2θ) and peak intensities.

111

10 20 30 40 50 60

0

10000

20000

30000

40000

50000

60000

70000

80000

Inte

nsity

2 (degree)

a

10 20 30 40 50 60

0

10000

20000

30000

40000

50000

60000

70000

80000

Inte

nsity

2 (degree)

b

10 20 30 40 50 60

0

10000

20000

30000

40000

50000

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Fig. 3 – PXRD patterns of DRO (a), β-CD (b), HP-β-CD (c), physical mixture with β-

CD (d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with β-CD (f)

and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by kneading

following spray-drying with HP-β-CD (j) and β-CD (k).

The PXRD pattern of DRO showed peaks with higher intensities at (2θ) 7.68,

8.10, 11.87, 13.00, 13.83, 15.71, 16.22, 19.96, 21.41, 21.60, 26.11, 27.56, attributed

to a crystalline state, with an ordered arrangement of the molecules. β-CD was also

in a crystalline form, with peaks with higher intensity at (2θ) 10.72, 12. 2, 1 .46,

17.11, 17.71, 18.99, 19.59, 22.72, 31.98, 34.78. On the other hand, the PXRD

pattern of HP-β-CD showed a broad peak characteristic of the amorphous state, in

line with previous reports [36,37].

The physical mixtures PXRD pattern showed the sum of individual peaks of

the substances with a higher degree in crystallinity when compared to the PXRD

patterns of the inclusion complexes.

From the analysis of the diagrams of the inclusion complexes with β-CD

obtained through lyophilization and colyophilization, it could be seen similar patterns,

probably because they were prepared by similar techniques. The diagrams of both

complexes presented a reduction in peak relative intensities and do not showed the

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114

characteristic diffraction pattern of DRO. The complexes obtained by lyophilization

presented peaks with high intensity at (2θ) 12.44, 17.06, 19. 7, 19.94, 20.72, most

of them attributed to β-CD. In relation to the inclusion complex obtained by kneading

following spray-drying (SB), the crystallinity loss was observed in the PXRD analysis,

confirming the phenomena observed in the DSC curve: the disappearance of DRO

melting point.

The PXRD patterns of inclusion complexes with HP-β-CD obtained by

lyophilization, colyophilization and spray-drying are very similar to the isolated HP-β-

CD diagram. These diffractograms do not present the pattern of the pure DRO,

showing the complete amorphization of the drug and the formation a new solid

phase, corroborating with the results obtained by the DSC analysis. In relation to the

preparation methods, it was evidenced that the kneading following spray-drying

technique provided the higher degree of amorphization.

3.3.4 Fourier-transform infrared spectroscopy (FT-IR)

Fourier-transform infrared spectroscopy was used in order to verify the

formation of the inclusion complexes, observed through vibrational changes upon

host:guest interactions between DRO and the CDs [38]. FT-IR spectra of samples

are shown in Fig. 4. DRO spectrum presented the main bands at 3448 cm-1 (referred

to N-H stretching from aromatic amide), 2959 cm-1 (C-H aromatic stretching), 1638

cm-1 (C=O stretching), 1334 and 1156 cm-1 (SO2 stretching) [39]. The β-CD and HP-

β-CD spectra present a broad and strong band at 3350 cm-1 (referred to stretching

vibrations of OH group) relative to intermolecular hydrogen bonds, a peak at 2900

cm-1 (relative to C-H aliphatic stretching vibration), a peak in the region of 1600 cm-1

115

(corresponding to C-O stretching vibrations in ester and deformation vibration of O-H

bonds), and bands in the region of 1150 and 1030 cm-1 (for C-O-C stretching

vibrations) [17,37,40].

The spectrum of the physical mixture with β-CD, by comparing with the

spectra of the pure drug and β-CD, showed peaks at the region of 3350, 1650 and

1031 cm-1, similar as those presented in the β-CD spectrum and a peak at 1339 cm-

1(SO2 stretching) attributed to DRO. The other DRO characteristic –NH stretching

band in the region around 3450 cm-1 was masked by the intense band characteristic

of CD in the range of 3500-3000 cm-1 (stretching vibration of OH). The same effect

was observed for the DRO intrinsic bands 2959 cm-1, 1638 cm-1 and 1156 cm-1 with

the characteristic β-CD bands in the ranges 2920-2933 cm-1, 1640-1660 cm-1 and

1158 cm-1. For this reason, probably due to the low DRO molecular ratio in relation to

CD (1:10), only the DRO band at 1334 cm-1 was used to analyze the inclusion

complexes.

The spectra of the inclusion complexes with β-CD and HP-β-CD revealed

modifications in vibration transitions in the bands related to DRO, which were defined

and narrow in DRO spectrum. In the spectra of inclusion complexes with β-CD, the

band in the region of 1334 cm-1 (related to S=O stretching) assigned to the pure drug

was changed to 1340 cm-1, however with lower intensity than in the physical mixture;

the decrease in intensity were higher for the complexes prepared by colyophilization

than lyophilization.

The alterations observed in the inclusion complexes with HP-β-CD were

similar with those previously described as the reduction and alteration in the peak

format at 1334 cm-1 in relation to the physical mixture. The wave number shifted to

1341, 1331 and 1338 cm-1 for complexes prepared by lyophilization, colyophilization

116

and spray-drying techniques, respectively. A higher reduction in peak intensity was

found for the inclusion complex obtained through spray-drying, followed by the one

obtained through colyophilization. Reduction and shift of the bands were also

reported by other authors as an evidence of complexation with β-CD and HP-β-CD

[19,41]. These events suggested an interaction between the drug and the

cyclodextrins, with the formation of a new crystalline phase, confirming the results

obtained by DSC and PXRD, probably as a consequence of inclusion complex

formation.

117

a

b

c

d

118

g

h

e

f

119

Fig. 4 – FT-IR spectra of DRO (a), β-CD (b), HP-β-CD (c), physical mixture with β-CD

(d) and HP-β-CD (e), inclusion complexes obtained by lyophilization with β-CD (f)

and HP-β-CD (g), by colyophilization with β-CD (h) and HP-β-CD (i) and by kneading

following spray-drying with HP-β-CD (j) and β-CD (k).

i

j

k

120

3.3.5 Scanning electron microscopy (SEM)

In order to support DSC, PXRD and FTIR analyses, SEM was used to

examine the surface morphology of pure dronedarone and its inclusion complexes

(Fig 5). DRO showed a crystalline form characterized by a rectangular shape and

size from 10 -100 µm, similar to the morphology described in a previous study [9]. β-

CD appeared as a large polyhedral form and HP-β-CD showed spherical particles.

The physical mixtures with β-CD and HP-β-CD showed the small drug crystals

dispersed among the CD particles. The inclusion complexes with β-CD and HP-β-CD

by lyophilization and colyophilization appear as irregular blocks granules, which are

different from the original components in morphology, confirming that a new solid

phase was formed by those techniques. This was also observed in inclusion

complexes of another poor water solubility drug with β-CD and HP-β-CD [19].

Following inclusion complexation of DRO and β-CD and HP-β-CD by kneading and

spray-drying, a drastic change in morphology was observed, with a great reduction in

particle size by forming homogenous spherical particles, joined to form

agglomerates. This change in morphology of spray dryer samples was also reported

for an inclusion complex between HP-β-CD and a poorly water soluble drug [42].

Finally, the SEM analysis taken together with the DSC, PXRD and FTIR

results, suggested the interaction between DRO and the CDs, indicating the

formation of amorphous complexes of DRO and β-CD and HP-β-CD.

121

Fig. 5 – SEM micrographs of DRO (a), β-CD (b), HP-β-CD (c), physical mixtures with

β-CD (d) and HP-β-CD (e), inclusion complexes obtained by colyophilization with β-

CD (f) and HP-β-CD (g), by lyophilization with β-CD (h) and HP-β-CD (i), and by

spray drying with β-CD (j) and HP-β-CD (k) presented at different magnification ((a)

DRO, 1000×; (b) and (c), 100×; (d) and (e), 500× and inclusion complexes, 200×).

3.4. Determination of aqueous solubility of DRO after complexation

The water solubilities of pure DRO and inclusion complexes were determined

by dissolving an excess of pure DRO and inclusion complexes in water, and kept

stirring at room temperature. After 24 h, samples were analyzed by the HPLC method

to determine the drug concentration. Pure DRO was practically insoluble in water; the

solubility was 0.73±0.17 mg mL -1 (mean± standard deviation). After the complexation

with β-CD and HP-β-CD, the water solubility of DRO increased to approximately 3

mg mL -1 (3.23±0.30 mg mL -1 for LB; 3.25±0.26 mg mL -1 for RB; 2.82±0.05 mg mL -1

for SB; 2.55±0.05 mg mL -1 for RH; and 2.69±0.41 mg mL -1 for SH). The about 4.0-

fold higher DRO solubility of the inclusion complexes confirmed that both CD

improved the water solubility of DRO, being this system a valuable product for the

122

development of novel drug delivery systems.

3.5. Dissolution studies

The dissolution rates of free DRO and the inclusion complexes in simulated

gastric fluid (pH 1.2), acetate buffer (pH 4.5) and phosphate buffer (pH 6.8) are

shown in Fig. 6. The dissolution profiles obtained in simulated gastric fluid (Fig. 6a)

for the inclusion complexes exhibited nearly 100% drug release. Statistical analyses

were performed using ANOVA following Dunnett’s multiple comparison test with the

percentages of DRO dissolved at 120 min and it was evidenced that all inclusion

complexes improved DRO dissolution rate significantly (p < 0.05). The inclusion

complex with β-CD prepared by kneading and spray-dryer showed the highest

amount of dissolved drug, with 100.06% (SD = 2.36) in 120 min. In fact, the amount

of drug dissolved in simulated gastric fluid increased approximately 4.5-fold after

complexation with the two CDs.

In the case of acetate pH 4.5, approximately 70% of DRO was dissolved in the

first 5 min of the experiment, while the dissolution was completed for all the inclusion

complexes with β-CD and HP-β-CD, except for that with HP-β-CD obtained by

lyophilization, with an initial release of 81.55%. The dissolution rate of the free drug

was closer to the dissolution rate of the inclusion complexes in pH 4.5 because DRO

solubility is higher in a weak acidic medium (pH 3 to 5) [10].

Fig. 6c shows the dissolution profiles plotted from the experimental values of

DRO and the inclusion complexes with β-CD and HP-β-CD in phosphate pH 6.8. The

free drug showed a release of only approximately 6% after 120 min. In contrast, the

releases from the inclusion complexes were around 60% until the same time.

123

Statistical analysis of the release rates in phosphate pH 6.8 after 120 min using

ANOVA following Dunnett’s multiple comparison test revealed a significant

improvement in DRO dissolution rate after complexation with CDs (p < 0.05). The

dissolution profiles also suggested a small difference in the release rates for the

inclusion complexes with β–CD obtained by lyophilization and colyophilization, with

53 and 54%. This behavior could be related to their respective PXRD characteristics,

in contrast with the others inclusion complexes, which presented a higher degree of

amorphization. However, this difference was not evidenced using Tukey’s multiple

comparison test (p > 0.05).

In line with our experimental data, and as evidenced in previous studies, a fast

dissolution is observed when a binary system drug-CD is dispersed in a dissolution

medium [43]. Considering that DRO is a poorly-water soluble drug, showing a pH-

dependent solubility profile, it is a relevant point to improve its solubility regardless

the pH of the GI tract. Then, according to the dissolution profiles of the inclusion

complexes with β-CD and HP-β-CD obtained through different methods, in different

pH values (1.2, 4.5 and 6.8), it was evidenced that the CDs markedly improved the

dissolution rate of DRO. As the percent drug release of the inclusion complexes were

higher than the pure drug in the three pH conditions, it was suggested that the

complexes have a great tendency to be well dissolved in the human GI tract [44].

124

Fig. 6 – Dissolution profiles of free DRO and inclusion complexes obtained by

lyophilization with β-CD (LB) and HP-β-CD (LH), by colyophilization with β-CD (RB)

and HP-β-CD (RH), and by kneading and spray drying with β-CD (SB) and HP-β- CD

(SH) in pH 1.2 (a), 4.5 (b) and 6.8 (c).

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3.6. Chemical stability

The inclusion complexes of dronedarone with β-CD and HP-β-CD were stored

in a climate stability chamber at 40°C and 75% relative humidity, simulating

accelerated storage conditions, and into the desiccator at room temperature for 30

days (D30). The content was determined by the HPLC method and the results are

shown in Table 2. The mean reduction in the content was about 7.5% (7.2% for LB,

14.4% for LH, 0.4% for RB, 6.7% for RH, 9.9% for SB and 6.1% for SH) for the

samples kept in stability chamber and 3.8% (9.7% for LB, 3.5% for LH, 2.7% for RB,

1.2% for RH, 2.0% for SB and 3.8% for SH) for samples kept in room temperature.

For the evaluation of new drug products under accelerated storage conditions, the

international guideline [45] recommends a 5% change in assay from its initial value.

Considering the assay results for the inclusion complexes, it was found that they not

meet the criteria, as the assay changes were higher than 5%, indicating a possible

reduced shelf-life, suggesting the need of an adequate container closure system to

protect the drug from humidity and heat.

The samples were also analyzed by PXRD and the diffraction patterns were

obtained (supplementary figures 1 and 2). The diffraction patterns of the inclusion

complexes revealed the presence of crystalline peaks, most intense in the complexes

stored in the stability chamber, demonstrating an increment in the crystallinity of the

samples. However, by comparing their diffraction patters with physical mixtures with

β-CD (Fig. 3d) and HP-β-CD (Fig. 3e), all the complexes stored in the different

conditions still present peaks with drastically reduced intensity. These results may

indicate that the interaction of DRO with CDs still remain until 30 days, however, the

inclusion complexes failed the acceptance criteria for assay during the accelerated

126

storage conditions.

3.7. In vitro cytotoxicity assay

The effects of free DRO and its inclusion complexes on the 3T3 cells viability

were evaluated using the MTT assay, and were illustrated in Fig. 7. The DRO

concentrations of 1.25, 2.5 and 5.0 µg mL-1 were chosen considering a previous

study [46], where it was suggested that DRO concentrations above 10 µM (5.932 µg

mL-1) could compromise the mitochondrial function.

As the Fig. 7 evidenced, the inclusion complexes exhibited a lower reduction

in cell viability in comparison to the free drug, especially with the concentration value

of 5.0 µg mL-1. Statistical analysis using ANOVA followed by the Dunnett’s multiple

comparison test (using the cell viability of the free drug as control) showed a

significant difference (p < 0.05) of the cytotoxicity of free DRO and the inclusion

complexes obtained by colyophilization and kneading followed by spray-drying with

β-CD and HP-β-CD, and by lyophilization with β-CD. In the concentration level of

1.25 mL-1, no sample induced cytotoxic effects on 3T3 cells.

The lipophilic compounds present more cytotoxic effects in relation to the

hydrophilic ones, which could be associated with modifications on cell membrane

structure induced by the first compounds [47]. In a cytotoxicity study of inclusion

complexes of a lipophilic drug with CD on Balb/c mice peritoneal macrophages [48],

a direct relation between cell toxicity and lipophilicity was made, attributed to the

higher degree of penetration into cell membranes of lipophilic substances.

Considering the results obtained by the in vitro cytotoxic assay in 3T3 cells, it could

be suggested that the reduction on DRO cytotoxicity when complex with CD, could

127

be attributed to the higher hydrophilicity/solubility of the inclusion complexes.

After oral administration of 400 mg of DRO twice daily, plasma concentrations

reached 84 to 167 ng/mL in the steady-state [11]. The concentration of 5.0 µg mL-1,

which was observed a cytotoxic effect, is almost 30 times higher than the plasmatic

concentration, suggesting that after oral administration, the cytotoxic concentrations

would not be reached.

Fig. 7 – Cell viability of free DRO and inclusion complexes with β-CD and HP-β-CD

obtained through different techniques on 3T3 cells, determined by the MTT assay.

Assay concentrations (left to right): 1.25 µg mL-1 (white), 2.5 µg mL-1 (gray) and 5.0

µg mL-1 (black). Statistical analyses were performed using ANOVA followed by

Dunnett’s multiple comparison test. * Statistically different (p < 0.05) using the free

drug as control.

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Free drug LB LH RB RH SB SH

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* * *

* *

128

4. Conclusions

In the present work, inclusion complexes of DRO with β-CD and HP-β-CD

were prepared by three different techniques and characterized by FT-IR, SEM, DSC

and PXRD, which suggested that the complexes obtained by kneading following

spray-drying were transformed to amorphous forms. Furthermore, the results showed

that the complexes showed better water solubility and faster dissolution rate in

relation to the pure drug. The in vitro cytotoxicity study indicated a reduction on the

cytotoxic effect of DRO upon complexation with CDs. Finally, these systems could be

promising approaches for the design of novel formulations containing DRO.

Considering the acceptable material properties of the inclusion complexes prepared

by kneading and spray-drying method, with reduced particle size and high shape

uniformity, it may be possible to prepare solid dosage forms such as tablets, by direct

compression process.

Acknowledgements

This work was supported by the Brazilian National Council for Scientific and

Technological Development (CNPq) [grant numbers 401069/2014-1 and

447548/2014-0]; FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio

Grande do Sul) [grant number 2293-2551/14-0]; and CAPES (Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior).

Prof. Dr. Gustavo Luiz Callegari (LMMM/CCNE/ Federal University of Santa Maria) is

acknowledged for his collaboration for XRD measurements.

129

References

[1] V. Fuster, L.E. Rydén, D.S. Cannom, H.J. Crijns, A.B. Curtis, K. a. Ellenbogen,

J.L. Halperin, J.Y. Le Heuzey, G.N. Kay, J.E. Lowe, S.B. Olsson, E.N.

Prystowsky, J.L. Tamargo, S. Wann, S.G. Priori, J.J. Blanc, A. Budaj, a. J.

Camm, V. Dean, J.W. Deckers, C. Despres, K. Dickstein, J. Lekakis, K.

McGregor, M. Metra, J. Morais, A. Osterspey, J.L. Zamorano, S.C. Smith, A.K.

Jacobs, C.D. Adams, J.L. Anderson, E.M. Antman, S.A. Hunt, R. Nishimura,

J.P. Ornato, R.L. Page, B. Riegel, ACC/AHA/ESC 2006 guidelines for the

management of patients with atrial fibrillation-executive summary: A report of

the American College of Cardiology/American Heart Association Task Force on

practice guidelines and the European Society of Cardiology Commi, Eur. Heart

J. 27 (2006) 1979–2030. doi:10.1093/eurheartj/ehl176.

[2] B.N. Singh, E. Aliot, Newer antiarrhythmic agents for maintaining sinus rhythm

in atrial fibrillation  simplicity or complexity ?, 9 (2007) 17–25.

doi:10.1093/eurheartj/sum049.

[3] S.H. Hohnloser, H.J.G.M. Crijns, M. van Eickels, C. Gaudin, R.L. Page, C.

Torp-Pedersen, S.J. Connolly, Effect of dronedarone on cardiovascular events

in atrial fibrillation, N. Engl. J. Med. 360 (2009) 668–678.

doi:10.1056/NEJMoa0803778.

[4] J.-Y. Le Heuzey, G.M. De Ferrari, D. Radzik, M. Santini, J. Zhu, J.-M. Davy, A

short-term, randomized, double-blind, parallel-group study to evaluate the

efficacy and safety of dronedarone versus amiodarone in patients with

persistent atrial fibrillation: The DIONYSOS Study, J. Cardiovasc.

Electrophysiol. 21 (2010) 597–605. doi:10.1111/j.1540-8167.2010.01764.x.

[5] W. Sun, J.S.M. Sarma, B.N. Singh, Chronic and acute effects of dronedarone

130

on the action potential of rabbit atrial muscle preparations: comparison with

amiodarone., J. Cardiovasc. Pharmacol. 39 (2002) 677–684.

doi:10.1097/00005344-200205000-00008.

[6] U.S. Food and Drug Administration, FDA labeling information: Revised Multaq

label, (2014).

http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/022425s025lbl.pdf

(accessed December 11, 2016).

[7] S.J. Connolly, A.J. Camm, J.L. Halperin, C. Joyner, M. Alings, J. Amerena, D.

Atar, Á. Avezum, P. Blomström, M. Borggrefe, A. Budaj, S.-A. Chen, C.K.

Ching, P. Commerford, A. Dans, J.-M. Davy, E. Delacrétaz, G. Di Pasquale, R.

Diaz, P. Dorian, G. Flaker, S. Golitsyn, A. Gonzalez-Hermosillo, C.B. Granger,

H. Heidbüchel, J. Kautzner, J.S. Kim, F. Lanas, B.S. Lewis, J.L. Merino, C.

Morillo, J. Murin, C. Narasimhan, E. Paolasso, A. Parkhomenko, N.S. Peters,

K.-H. Sim, M.K. Stiles, S. Tanomsup, L. Toivonen, J. Tomcsányi, C. Torp-

Pedersen, H.-F. Tse, P. Vardas, D. Vinereanu, D. Xavier, J. Zhu, J.-R. Zhu, L.

Baret-Cormel, E. Weinling, C. Staiger, S. Yusuf, S. Chrolavicius, R. Afzal, S.H.

Hohnloser, Dronedarone in High-Risk Permanent Atrial Fibrillation, N. Engl. J.

Med. 365 (2011) 2268–2276. doi:10.1056/NEJMoa1109867.

[8] T.G.A. Australian Government, Department of Health and Ageing, Australian

Public Assessment Report for Dronedarone Hydrochloride, (2010) 1–115.

https://www.tga.gov.au/sites/default/files/auspar-multaq.pdf (accessed January

6, 2017).

[9] S.D. Han, S.W. Jung, S.W. Jang, H.J. Jung, M. Son, B.M. Kim, M.J. Kang,

Preparation of Solid Dispersion of Dronedarone Hydrochloride with Soluplus ®

by Hot Melt Extrusion Technique for Enhanced Drug Release, Chem. Pharm.

131

Bull. 63 (2015) 295–299.

[10] S.D. Han, S.W. Jung, S.W. Jang, M. Son, B.M. Kim, M.J. Kang, Reduced

Food-Effect on Intestinal Absorption of Dronedarone by Self-microemulsifying

Drug Delivery System (SMEDDS), Biol. Pharm. Bull. 38 (2015) 1026–1032.

doi:10.1248/bpb.b15-00110.

[11] C. Patel, G.-X. Yan, P.R. Kowey, Dronedarone, Circulation. 120 (2009) 636–

644. doi:10.1161/CIRCULATIONAHA.109.858027.

[12] Z. Fülöp, P. Saokham, T. Loftsson, Sulfobutylether-β-cyclodextrin/chitosan

nano- and microparticles and their physicochemical characteristics, Int. J.

Pharm. 472 (2014) 282–287. doi:10.1016/j.ijpharm.2014.06.039.

[13] S.S. Jambhekar, P. Breen, Cyclodextrins in pharmaceutical formulations I:

Structure and physicochemical properties, formation of complexes, and types

of complex, Drug Discov. Today. 21 (2016) 356–362.

doi:10.1016/j.drudis.2015.11.017.

[14] T. Loftsson, D. Duchêne, Cyclodextrins and their pharmaceutical applications,

Int. J. Pharm. 329 (2007) 1–11. doi:10.1016/j.ijpharm.2006.10.044.

[15] C. Mendes, A. Buttchevitz, J.H. Kruger, J.M. Kratz, C.M.O. Sim?es, P. de

Oliveira Benedet, P.R. Oliveira, M.A.S. Silva, Inclusion complexes of

hydrochlorothiazide and β-cyclodextrin: Physicochemical characteristics, in

vitro and in vivo studies, Eur. J. Pharm. Sci. 83 (2016) 71–78.

doi:10.1016/j.ejps.2015.12.015.

[16] P. Mura, Analytical techniques for characterization of cyclodextrin complexes in

aqueous solution  A review, J. Pharm. Biomed. Anal. 101 (2014) 2 8–250.

doi:10.1016/j.jpba.2014.02.022.

[17] X. Wang, Z. Luo, Z. Xiao, Preparation, characterization, and thermal stability of

132

beta-cyclodextrin/soybean lecithin inclusion complex, Carbohydr Polym. 101

(2014) 1027–1032. doi:10.1016/j.carbpol.2013.10.042.

[18] H. Liu, G. Yang, Y. Tang, D. Cao, T. Qi, Y. Qi, G. Fan, Physicochemical

characterization and pharmacokinetics evaluation of beta-caryophyllene/beta-

cyclodextrin inclusion complex, Int. J. Pharm. 450 (2013) 304–310.

doi:10.1016/j.ijpharm.2013.04.013.

[19] P. Tang, S. Li, L. Wang, H. Yang, J. Yan, H. Li, Inclusion complexes of

chlorzoxazone with β- and hydroxypropyl-β-cyclodextrin: Characterization,

dissolution, and cytotoxicity, Carbohydr. Polym. 131 (2015) 297–305.

doi:10.1016/j.carbpol.2015.05.055.

[20] S. V. Kurkov, T. Loftsson, Cyclodextrins, Int. J. Pharm. 453 (2013) 167–180.

doi:10.1016/j.ijpharm.2012.06.055.

[21] T. Higuchi, K.A. Connors, Phase solubility techniques, in: C.N. Reilly (Ed.),

Adv. Anal. Chem. Instrum., Interscience Publishers, New York, N.Y., 1965: pp.

117–212.

[22] T. Loftsson, D. Hreinsdóttir, M. Másson, The complexation efficiency, J. Incl.

Phenom. Macrocycl. Chem. 57 (2007) 545–552. doi:10.1007/s10847-006-

9247-2.

[23] D.R. Nogueira, M. Mitjans, M.R. Infante, M.P. Vinardell, Comparative sensitivity

of tumor and non-tumor cell lines as a reliable approach for in vitro cytotoxicity

screening of lysine-based surfactants with potential pharmaceutical

applications, Int. J. Pharm. 420 (2011) 51–58.

doi:10.1016/j.ijpharm.2011.08.020.

[24] C.S. Paim, D.R. Nogueira, M. Mitjans, D. Ramos Lopez, J. de Lapuente Perez,

M. Steppe, E.E.S. Schapoval, M.P. Vinardell, Biological safety studies of

133

gemifloxacin mesylate and related substances, Photochem. Photobiol. Sci. 12

(2013) 805–812. doi:10.1039/c3pp25369d.

[25] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays., J. Immunol. Methods. 65

(1983) 55–63.

[26] M. Kfoury, L. Auezova, H. Greige-Gerges, S. Fourmentin, Development of a

Total Organic Carbon method for the quantitative determination of solubility

enhancement by cyclodextrins  Application to essential oils, Anal. Chim. Acta.

918 (2016) 21–25. doi:10.1016/j.aca.2016.03.013.

[27] T. Loftsson, D. Hreinsdóttir, M. Másson, Evaluation of cyclodextrin

solubilization of drugs, Int. J. Pharm. 302 (2005) 18–28.

doi:10.1016/j.ijpharm.2005.05.042.

[28] L.A. Miller, R.L. Carrier, I. Ahmed, Practical considerations in development of

solid dosage forms that contain cyclodextrin, J. Pharm. Sci. 96 (2007) 1691–

1707. doi:10.1002/jps.20831.

[29] M.E. Brewster, T. Loftsson, Cyclodextrins as pharmaceutical solubilizers, Adv.

Drug Deliv. Rev. 59 (2007) 645–666. doi:10.1016/j.addr.2007.05.012.

[30] S.K. Singh, P.R. Vuddanda, S. Singh, A.K. Srivastava, A Comparison between

Use of Spray and Freeze Drying Techniques for Preparation of Solid Self-

Microemulsifying Formulation of Valsartan and In Vitro and In Vivo Evaluation,

Biomed Res. Int. 2013 (2013) 1–13. doi:10.1155/2013/909045.

[31] F. Giordano, C. Novak, J.R. Moyano, Thermal analysis of cyclodextrins and

their inclusion compounds, Thermochim. Acta. 380 (2001) 123–151.

doi:10.1016/S0040-6031(01)00665-7.

[32] L.J. Yang, S. Xia, S.X. Ma, S.Y. Zhou, X.Q. Zhao, S.H. Wang, M.Y. Li, X.D.

134

Yang, Host-guest system of hesperetin and β-cyclodextrin or its derivatives:

Preparation, characterization, inclusion mode, solubilization and stability,

Mater. Sci. Eng. C. 59 (2016) 1016–1024. doi:10.1016/j.msec.2015.10.037.

[33] A. Zou, X. Zhao, U.A. Handge, V.M. Garamus, R. Willumeit-Römer, P. Yin,

Folate receptor targeted bufalin β-cyclodextrin supramolecular inclusion

complex for enhanced solubility and anti-tumor efficiency of bufalin, Mater. Sci.

Eng. C. 78 (2017) 609–618. doi:10.1016/j.msec.2017.04.094.

[34] P. Mura, Analytical techniques for characterization of cyclodextrin complexes in

the solid state: A review, J. Pharm. Biomed. Anal. 113 (2015) 226–238.

doi:10.1016/j.jpba.2015.01.058.

[35] A.I. Takahashi, F.J.B. Veiga, H.G. Ferraz, A literature review of cyclodextrin

inclusion complexes characterization - Part III  differential scanning calorimetry

and thermogravimetry, Int. J. Pharm. Sci. Rev. Res. 12 (2012) 16–20.

[36] S.X. Ma, W. Chen, X.D. Yang, N. Zhang, S.J. Wang, L. Liu, L.J. Yang,

Alpinetin/hydroxypropyl-β-cyclodextrin host-guest system: Preparation,

characterization, inclusion mode, solubilization and stability, J. Pharm. Biomed.

Anal. 67–68 (2012) 193–200. doi:10.1016/j.jpba.2012.04.038.

[37] N. Qiu, X. Cheng, G. Wang, W. Wang, J. Wen, Y. Zhang, H. Song, L. Ma, Y.

Wei, A. Peng, L. Chen, Inclusion complex of barbigerone with hydroxypropyl- ␤

-cyclodextrin  Preparation and in vitro evaluation, Carbohydr. Polym. 101

(2014) 623–630. doi:10.1016/j.carbpol.2013.09.035.

[38] G. Yurtdaş, M. Demirel, L. Genç, Inclusion complexes of fluconazole with β-

cyclodextrin: Physicochemical characterization and in vitro evaluation of its

formulation, J. Incl. Phenom. Macrocycl. Chem. 70 (2011) 429–435.

doi:10.1007/s10847-010-9908-z.

135

[39] M. Mahender, M. Saravanan, C. Sridhar, E.R.R. Chandrashekar, L.J. Kumar,

A. Jayashree, R. Bandichhor, Identification and Characterization of Potential

Impurities of Dronedarone Hydrochloride, Org. Process Res. Dev. 18 (2014)

157–162.

[40] M.K. Riekes, M.P. Tagliari, A. Granada, G. Kuminek, M.A.S. Silva, H.K.

Stulzer, Enhanced solubility and dissolution rate of amiodarone by

complexation with β-cyclodextrin through different methods, Mater. Sci. Eng. C.

30 (2010) 1008–1013. doi:10.1016/j.msec.2010.05.001.

[41] A.I. Takahashi, F. José, B. Veiga, H.G. Ferraz, A literature review of

cyclodextrin inclusion complexes characterization - Part II: X-ray diffraction,

infrared spectroscopy and nuclear magnetic resonance, Int. J. Pharm. Sci. Rev.

Res. 12 (2012) 8–15.

[42] M. Shah, Y. Pore, S. Dhawale, K. Burade, B. Kuchekar, Physicochemical

characterization of spray dried ternary micro-complexes of cefuroxime axetil

with hydroxypropyl-β-cyclodextrin, J. Incl. Phenom. Macrocycl. Chem. 76

(2013) 391–401. doi:10.1007/s10847-012-0211-z.

[43] N.B. Naidu, K.P.R. Chowdary, K.V.R. Murthy, V. Satyanarayana, A.R. Hayman,

G. Becket, Physicochemical characterization and dissolution properties of

meloxicam–cyclodextrin binary systems, J. Pharm. Biomed. Anal. 35 (2004)

75–86. doi:10.1016/j.jpba.2004.01.003.

[44] P. Tang, Q. Wang, K. Xu, L. Wang, S. Li, H. Li, Investigation the complex of

2,6-di-O-methyl-β-cyclodextrin and chlorzoxazone: Preparation,

characterization, dissolution, and cytotoxicity, J. Incl. Phenom. Macrocycl.

Chem. 83 (2015) 167–176. doi:10.1007/s10847-015-0556-1.

[45] ICH Q1A (R2), Stability Testing of New Drug Substances and Products

136

Q1A(R2), in: Int. Conf. Harmon., 2003.

[46] A. Felser, K. Blum, P.W. Lindinger, J. Bouitbir, S. Krähenbühl, Mechanisms of

hepatocellular toxicity associated with dronedarone - A comparison to

amiodarone, Toxicol. Sci. 131 (2013) 480–490. doi:10.1093/toxsci/kfs298.

[47] A.O. De Souza, R.R. Santos-jr, D.N. Sato, M.M.M. De Azevedo, A. Denise,

P.S. Melo, M. Haun, C.L. Silva, N. Durán, Free 2-Propen-1-amine Derivative

and Inclusion Complexes with β-Cyclodextrin: Scanning Electron Microscopy,

Dissolution, Cytotoxicity and Antimycobacterial Activity, J. Braz. Chem. Soc. 15

(2004) 682–689.

[48] M. a M. Lyra, J.L. Soares-Sobrinho, R.C.B.Q. Figueiredo, J.M. Sandes, Á. a N.

Lima, R.P. Tenório, D. a F. Fontes, F.L. a Santos, L. a. Rolim, P.J. Rolim-Neto,

Study of benznidazole-cyclodextrin inclusion complexes, cytotoxicity and

trypanocidal activity, J. Incl. Phenom. Macrocycl. Chem. 73 (2012) 397–404.

doi:10.1007/s10847-011-0077-5.

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SUPPLEMENTARY FIGURES

Fig. 1 - PXRD patterns of inclusion complexes obtained by lyophilization with β-CD

(a), and HP-β-CD (b), by colyophilization with β-CD (c), and HP-β-CD (d) and by

kneading following spray-drying with HP-β-CD (e) and β-CD (f) after storage in

climate stability chamber for 30 days.

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Fig. 2 - PXRD patterns of inclusion complexes obtained by lyophilization with β-CD

(a), and HP-β-CD (b), by colyophilization with β-CD (c), and HP-β-CD (d) and by

kneading following spray-drying with HP-β-CD (e) and β-CD (f) after storage into

desiccator for 30 days.

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4 ARTIGO 3 – AVALIAÇÃO DO POTENCIAL HEPATOTÓXICO, FOTOTÓXICO E

FOTOSSENSIBILIZANTE DO CLORIDRATO DE DRONEDARONA E SEUS

COMPLEXOS DE INCLUSÃO COM CICLODEXTRINAS

Publicação científica: Marcolino, A.I.P; Nogueira-Librelotto, D.R.; Mitjans, M.;

Vinardell, M.P.; Rolim; C.M.B.. Evaluation of the hepatotoxic, phototoxic and

photosensitizing potential of dronedarone hydrochloride and its inclusion complexes

with cyclodextrins. Manuscrito em preparação.

142

INTRODUÇÃO

Nesse estudo, avaliou-se o potencial hepatotóxico, fototóxico e fotossensibilizante

do cloridrato de dronedarona e seus complexos de inclusão com β-ciclodextrina e 2-

hidroxipropil-β-ciclodextrina utilizando ensaios de citotoxicidade in vitro. Dentre esse

ensaios, foram realizados o teste de fototoxicidade in vitro 3T3 NRU, o fotoensaio

utilizando a linhagem celular de leucemia monocítica aguda humana (THP-1) e

liberação de interleucina-8 e o ensaio de citotoxicidade em células tumorais de

hepatoma humano (HepG2). O estudo foi desenvolvido no Departamento de

Fisiologia da Universidade de Barcelona, Barcelona, Espanha durante a realização

do Doutorado Sanduich no Exterior (SWE), pelo Programa Ciências sem Fronteiras

(CNPq), sob a orientação da Prof. Dra. María Pilar Vinardell Martínez-Hidalgo e da

Prof. Dra. Montserrat Mitjans.

143

.

144

Evaluation of the hepatotoxic, phototoxic and photosensitizing potential of

dronedarone hydrochloride and its inclusion complexes with cyclodextrins

Ana Isa Pedroso Marcolinoa, Daniele Rubert Nogueira-Librelottoa,b, Montserrat

Mitjansc, María Pilar Vinardellc and Clarice Madalena Bueno Rolima,b*

aPostgraduate Program in Pharmaceutical Sciences, Federal University of Santa

Maria, Av. Roraima 1000, 97105-900, Santa Maria – RS, Brazil

bDepartment of Industrial Pharmacy, Federal University of Santa Maria, Av. Roraima

1000, 97105-900, Santa Maria – RS, Brazil

cDepartment of Biochemistry and Physiology, Faculty of Pharmacy and Food

Science, University of Barcelona, Joan XXIII 27-31, 08028, Barcelona – Spain

* Corresponding author. Department of Industrial Pharmacy, Federal University of

Santa Maria, Santa Maria – RS 97015-900, Brazil. Tel.: (+55) 55 3220 8645. Fax:

(+55) 55 3220 8248.

E-mail address: [email protected] (C.M.B. Rolim).


145

Abstract

In this study, the phototoxicity, hepatotoxicity and photosensitizing potential of

free dronedarone and its inclusion complexes with β-CD and HP-β-CD were

investigated by using in vitro cell-based approaches. The results of the 3T3 NRU

phototoxicity assay showed that free dronedarone and the inclusion complexes did

not present phototoxic potential. However, an exception was the inclusion complex

with HP-β-CD prepared through colyophilization, which presented a minor phototoxic

effect. The photosensitization was assessed by using THP-1 cells and IL-8 as a

biomarker, and the experimental data evidenced that both the free drug and inclusion

complexes showed potential to cause skin sensitization, as they were able to induce

IL-8 release after irradiation. Nevertheless, the inclusion complex with β-CD obtained

by kneading following spray-drying induced a significant lower release of IL-8 and

also presented the lowest stimulation index in comparison with free dronedarone,

suggesting a reduction in the photosensitizing potential. The free drug and inclusion

complexes were also tested for hepatotoxicity by using HepG2 cells. Even though

lower IC50 values were found for the inclusion complexes prepared by kneading

following spray-drying, there was no significant difference, indicating that the

complexation did not alter the hepatotoxic potential of dronedarone. Overall, the data

suggest that dronedarone is not phototoxic, however, it presents photosensitizing

potential. The inclusion complex prepared by kneading following spray-dryer is

suggested as a formulation which might reduce the photoallergic potential of

dronedarone.

Keywords: Dronedarone. Cyclodextrins. Inclusion complex. Cytotoxicity. In vitro 3T3

NRU phototoxicity assay. Photosensitization. Hepatotoxicity.

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1. Introduction

Dronedarone (DRO) is a new antiarrhythmic agent indicated to reduce the

hospitalization rate in patients with atrial fibrillation. This benzofuran derivative was

obtained from modifications of amiodarone molecule with the intention to reduce its

adverse effects, by reducing its lipophilicity and then the accumulation in tissues

[1,2]. DRO is metabolized by cytochrome P450 3A4, and is also a moderate inhibitor

[3]. DRO is a biopharmaceutics classification system II compound with pH-dependent

aqueous solubility, practically insoluble at pH 7 [4,5]. Regardless its adverse effects,

hepatocellular liver injury, even requiring liver transplantation, has been reported in

the postmarket setting of DRO tablets [6]. A case of fatal lung toxicity was also

reported after DRO use [7]. Photosensitive reactions occurred in a patient taking

DRO for one month, showing the drug potential to cause a photodistributed drug

eruption, even though this reaction appeared to be uncommon, affecting 1% of the

patients [8].

Cyclodextrins (CDs) are pharmaceutical excipients of the family of cyclic

oligosaccharides. The natural α-, β- and γ-CDs are formed by 6, 7 and 8 (α-1,4-)-

linked D-glucopyranose units, which have limited aqueous solubility. The CD

derivative 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) has been synthesized to

present higher water-solubility. The CD structure presents a lipophilic central cavity

and a hydrophilic outer surface. CDs form inclusion complexes like guest-host, where

the guests are hydrophobic drug moieties that are entrapped into the central cavity.

As a result of complexation, changes occur in the physicochemical properties of the

guest molecule, such as enhanced solubility and bioavailability of poor-water soluble

drugs [9–11]. Hydrophilic CDs like HP-β-CD are capable to enhance permeation of

lipophilic drugs or to reduce drug permeation through lipophilic membranes by

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reducing the partition from the exterior to the membrane, and could also increase

drug chemical stability at the aqueous membrane exterior [12]. In relation to safety

and toxicology of CDs, they are practically non-toxic after oral administration, as only

negligible amounts are able to permeate lipophilic membranes such as

gastrointestinal mucosa 1 . β-CD cannot be used in parenteral administration as it

can result in renal toxicity, in contrast, HP-β-CD are suitable and can be found in

market parenteral formulations [13,14]. The formation of inclusion complexes could

also be described as a micro-encapsulation process, as the guest molecule is

surrounded by the cyclodextrin molecules, altering the chemical, physical and

biological properties [15], such as stabilization against effects of light degradation

[16]; decreasing the biomass and cellular activity of Staphylococcus and toxicity

against leucocytes [17]; and enhancing anti-proliferative activity in cancer cells while

reducing cytotoxicity in normal lung fibroblast cells (MTC-5) [18]. Then, the purpose

of overcome certain limitations has stimulated the investigations into cyclodextrin

applications [19].

Safety is a primary concern when developing new pharmaceutical

formulations. Thus, toxicological issues of the drug formulation must be investigated

and approved according to available legislation procedures, before the intended use.

Phototoxic side effects of pharmaceutical formulations are of increasing concern,

urging the need of pre-clinical tests for side effects, particularly to detect phototoxic

potential of chemicals [20].

Photoreactions to pharmaceutical products are side effects that can be

triggered after exposure to environment light, mainly in response to UVA light (range

of 315-400 nm), which penetrates deep into epidermis and dermis, possessing

mutagenic and carcinogenic activity mediated by oxidative stress [21]. Drug induced

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photoirritancy (phototoxicity) is defined as tissue response following topical or

systemic administration of pharmaceutical substances. DRO presents a high

absorption in UV range, with maximum absorption peaks at 217 and 289 nm (with a

shoulder until 350 nm).

In contrast, photoallergy is an immunologically-mediated reaction to a

chemical, initiated by the formation of a photoproduct following a photochemical

reaction [20,22,23]. The mechanism of photoallergy is consider to be a form of

delayed type of hypersensitivity, being immunologically mediated [20]. The first stage

of the photosensitizing process is the absorption of photons of the appropriate

wavelength (ultraviolet or visible radiation) by the exogenous agent (photosensitizing

drug), that reach an excited state. The excited energy is transferred to oxygen

molecules, generating reactive oxygen species (ROS), which can induce local

oxidative stress and damages to genomic DNA, lipids and proteins in cells [22]. The

next step is the uptake of the photochemically converted exogenous agent (in

combination with carrier proteins, forming a complete antigen) by the antigen-

presenting cells, such as Langerhans cells present in the skin [20,24]. These cells

present the antigen to the lymph node, thereby inducing sensitization, and during this

phase, they differentiate and mature immunostimulatory cells by up-regulating the

expression of several co-stimulatory molecules and secreting various cytokines, such

as IL-8 [25].

In order to assess photosafety in vitro with a correlation with in vivo

observations, photosafety assays are conducted and cytotoxicity assays as in vitro

endpoints are explored, always taking into account the need of biological markers to

discriminate allergy and irritation without animal testing [23,25]. The most used assay

to evaluate the phototoxic potential of the drug is the 3T3 Neutral Red Uptake

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phototoxic test, the first alternative method accepted by OECD, replacing animal

testing [26,27]. The assay is focused on the effect of UVA light exposure on cell

viability, which is measured by the inhibition of the capacity of cell cultures to take up

the NR dye after a specific time in comparison to non-treated cells [23]. In this

validated study, amiodarone hydrochloride is described as phototoxic. In the case of

photosensitization and photoallergic reactions, the use of THP-1 cells and the IL-8

release was proposed as a model to identify the potential of chemicals to induce skin

sensitization [24].

Regarding investigations on liver toxicity, the mechanisms underlying DRO

hepatotoxicity were studied by using isolated rat liver mitochondria, primary human

hepatocytes and a well-characterized human hepatoma cell line HepG2. DRO was

described to inhibit transport chain and β-oxidation and uncoupling oxidative

phosphorylation of liver mitochondria, and the study associated this mechanism with

the liver injury reported in patients [28].

In this study, we focused on the safety status of DRO hydrochloride and its

inclusion complexes with β-CD and HP-β-CD and hypothesized that DRO toxicity

would be lower because of its complexation with CDs. The phototoxic potential of

DRO has not been described in the literature up this moment. In order to investigate

the mechanisms underlying the photochemical reactivity of DRO and its inclusion

complexes, two photosafety analytical studies were used: the 3T3 Neutral Red

Uptake phototoxicity test and a photoassay using a human cell line cultured in vitro

(THP-1 monocytes), considering the interleukin 8 (IL-8) expression as endpoint. In

addition, we aimed to investigate the hepatotoxic effects associated with DRO using

HepG2 cells, following by a comparison with those of the inclusion complexes with

CDs.

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2. Material and methods

2.1. Chemicals

DRO hydrochloride (purity> 98. %), β-CD and HP-β-CD were obtained from

Zibo Qianhui Biotechnology Co., Ltd. (Zibo, Shandong, China). Chlorpromazine

hydrochloride (CPZ), dimethyl sulfoxide (DMSO), 2,5-diphenyl-3,-(4,5-dimethyl-2-

thiazolyl) tetrazolium bromide (MTT), Neutral Red (NR) dye, 2-mercaptoethanol and

, ′, , ′-tetramethylbenzidine liquid substrate, supersensitive, for ELISA were

obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol was purchased from

Panreac (Barcelona, Spain). Trypsin-EDTA solution (0.5 g/L trypsin and 0.2 g/L

EDTA), phosphate buffered saline (PBS), fetal bovine serum (FBS) and Dulbecco’s

Modified Eagle’s Medium (DMEM), DMEM without phenol red, and RPMI-1640

medium, L-glutamine and antibiotic/ antimicotic (100 μg mL of streptomycin sulfate

and 100 U/mL potassium penicillin) were purchased from Lonza (Verviers, Belgium).

For all analyses, ultrapure water was purified with Millipore Milli-Q Plus Ultra-Pure

Water Purifier (Germany).

2.2. Cell culture

The murine fibroblast cell line NIH-3T3 and the human hepatoma cell line

HepG2 were maintained in DMEM (with 2 mM L-glutamine, 100 U/mL penicillin, 100

µg/mL streptomycin), supplemented with 10% (v/v) of heat inactivated FBS. The

human monocytic leukemia cell line THP-1 were cultured at 37°C and 5% CO2 in

RPMI-1640 medium containing 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL

streptomycin, 50 µM 2-mercaptoethanol, and supplemented with 10% (v/v) of heat

151

inactivated FBS. Cells were kept in a cell incubator with 5% CO2 at 37°C and were

harvested by trypsinization when reached around 80% confluence. The cell number

was determined using a Neubauer hemacytometer and the viability using the trypan

blue exclusion method.

2.3. Test compounds

2.3.1. Preparation of inclusion complexes

The solid inclusion complexes of DRO (molecular weight 9 .2 g mol) with β-

CD (molecular weight 1135.0 g/mol) and HP-β-CD (molecular weight 1540.0 g/mol)

were prepared by three different methods with a 1:10 molar ratio (drug: cyclodextrin).

Lyophilization. Stoichiometric amount of DRO and β-CD or HP-β-CD (1:10,

M/M) were mixed in a mortar for 10 min. The mixture was dissolved in water at 50°C.

Next, the pH of the suspension was adjusted to 4.5 with acetic acid and it was stirred

at room temperature for 24 h. The resulting suspension was frozen at -20°C with

lactose (10%, p/v) for 24 h and lyophilized for 48 h.

Colyophilization. Appropriate quantities of DRO and β-CD or HP-β-CD (1:10,

M/M) were dissolved in hydroalcoholic solution (1:1, v/v), kept in agitation for 24h.

Ethanol was then removed in a rotary evaporator at 50 ± 5ºC. The pH value was then

adjusted to 4.5 and lactose (10%, p/v) was added to the resulting suspension, which

was frozen and lyophilized.

Kneading and spray-drying. The powders of DRO and β-CD or HP-β-CD were

mixed in a mortar for 20 min. Then, 0.5 mL of water was added and mixed again for 5

min to form a paste, which was solubilized in 25 mL of water at 50°C for 20 min. The

pH of the suspension was adjusted to 4.5, following by stirring for 24 h at room

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temperature. The suspension was dried in a spray dryer model LM MSD 1.0

(Labmaq, Ribeirão Preto, SP, Brazil) with the following operation conditions: inlet

temperature: 120ºC, air pressure: 3 kgf/cm2, feed flow rate: 0.21L/h.

2.3.2. Preparation of sample solutions

The samples were freshly prepared and, accordingly to their solubility, the

stock solution of the free DRO was dissolved in methanol, while the solutions of the

inclusion complexes were prepared in ultrapure water. The stock solutions were

prepared at the concentration of 1 mg/mL DRO. The stock solution of chlorpromazine

was diluted in DMSO at the final concentration of 5 mg/mL.

2.4. Irradiation source

The plates were irradiated by using three UVA lamps Actinic BL TL/TL-D/T5

(Philips®, 43 V, 352 nm, 15 W) placed in a photostability chamber (58 × 34 × 28 cm).

Irradiance was checked with a photoradiometer Delta OHM equipped with a UVA

probe (HD2302- Italy), placed below the plate lid for accurate measurements.

Irradiance was determined to be 1.8 mW/cm².

2.5. In vitro cytotoxicity studies

2.5.1. 3T3 Neutral Red Uptake phototoxicity test

The 3T3 Neutral Red Uptake phototoxicity test was conducted according to

the OECD TG 432 guideline, with some modifications [26]. First, the fibroblast

sensitivity to radiation was tested in different doses of UVA (1.0, 1.7, 1.9, 2.5 and 5.0

153

J/cm²). The test was then performed with the UVA dose that provided > 80% cell

viability after irradiation. The NIH 3T3 murine fibroblast cell line was seeded in the

central 60 wells of 96-well cell culture plates (cell density of 1×105 cells/mL). After 24

h of incubation (5% CO2, 37°C), the cells were washed with 150 µL PBS and the

medium was replaced by 100 µL fresh DMEM (without phenol red) supplemented

with 5% FBS containing the free drug and the inclusion complexes in the

concentration range from 0.3 to 15.0 µg/mL. The plates were incubated (5% CO2,

37°C) in the dark for 60 min. Then, the selected plate was irradiated with an

irradiation dose of 1.7 J/cm² within 15 min of light exposure. In parallel, another plate

was prepared and kept in the dark, as a control (non-irradiated). At the end of the

exposure period, cells were washed with 150 µL PBS, the medium was replaced and

plates were incubated overnight (5% CO2, 37°C). Chlorpromazine was tested as UVA

positive control in the concentration range from 0.35 to 90 µg/mL.

2.5.2. Determination of the photosensitizing potential using THP-1 cells

The evaluation of the photosensitizing potential of DRO and the inclusion

complexes with CDs were performed according to a protocol used to identify

photoallergenic chemicals [24]. The THP-1 cells were seeded into 24-well plates at a

density of 1×106 cells/mL. Each well was filled with 500 µL of RPMI medium

supplemented with 10% FBS (v/v), where 5 µL of increasing drug concentration

(0.625, 1.25 and 2.50 µg/mL of free DRO and equivalent of inclusion complexes) or

vehicle (methanol, ultrapure water and DMSO) were added. CPZ, a known

photoallergen, was tested at 0.1 µg/mL. Immediately after applying the chemical

treatment, one plate was irradiated with UVA, in order to provide an irradiation dose

of 1.9 J/cm². A non-irradiated control plate was prepared in parallel and kept in the

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dark. After 24 h of incubation at 37 °C, plates were centrifuged at 1200 rpm for 5 min

in order to assess IL-8 release in the free supernatants, which were stored at -20°C

until analysis. Stimulation indexes (SI) were used to detect photoallergens and were

calculated as the ratio of IL-8 release for treated cells against untreated cells for

irradiated (I-SI) and non-irradiated cells (NI-SI). The ratio between these two indexes

(I-SI/NI-SI) was the overall stimulation index.

2.5.3. Cytotoxicity in HepG2 cells

HepG2 cells (1×105 cells/ mL) were grown in the central 60 wells of 96-well

cell culture plates in DMEM supplemented with 10 % FBS. After 24 h of incubation

(5% CO2, 37°C), the media was removed and the samples of free drug and inclusion

complexes were applied, prepared in DMEM containing 5% FBS and in the

concentration range from 0.3 to 15.0 µg/mL. Afterward, plates were incubated

overnight at 37°C in 5% CO2.

2.6. Cell viability assays

Cell viability of the in vitro phototoxicity assay were measured by the Neutral

Red Uptake test. Following treatment, cells were washed with 150 µL PBS and then

100 µL Neutral Red solution at 50 µg/mL were added at each well. After 3 h of

incubation (5% CO2, 37°C), cells were washed with 150 µL PBS and 150 µL of NR

desorb solution (water: ethanol: acetic acid; 49:50:1, v/v/v) was added.

Cytotoxicity in THP-1 and HepG2 cells were performed by the MTT test,

according to the method of Mosmann [29]. Cell viability was determined by the

percentage of tetrazolium salt reduction by viable cells against untreated cells. In the

155

photoassay using THP-1 cells, 500 µL of a MTT solution at 0.75 mg/mL were added

to each well. The plate was incubated for 3 h (5% CO2, 37°C), centrifuged and then

500 µL of acidified isopropanol was added to lyse the cells. Next, an aliquot of 100 µL

of each well was transferred to a 96-well plate. For each sample, the 75% viability

was calculated.

For the assessment of cytotoxicity in HepG2 cells, following overnight

incubation, 100 µL of MTT solution at 0.5 mg/mL were added to each well and after 3

h of incubation at 37°C in 5% CO2, the formazan product was dissolved with 100 µL

of DMSO.

In all in vitro cytotoxicity assays, after 10 min on a microtitre-plate shaker,

absorbance was read at 550 nm using a Tecan Sunrise microplate reader equipped

with Magellan (v. 6.6) software (Männedorf, Switzerland). Results were expressed as

the percentage of viability compared with control wells (the mean optical density of

untreated cells was set as 100 % viability).

2.7. IL-8 release measurements

Human interleukin-8 (IL-8) release from free supernatants was determined

using an enzyme-linked immunosorbent assays (ELISA) kit (BD OptEIA™) from BD

Biosciences (San Diego, CA, USA). Results are expressed in pg/mL.

Based on the release of IL-8 from cells treated with the respective

concentrations of the products, stimulation indexes (SI) were determined, according

to Martínez [24]. The stimulation indexes were calculated by the ratio of the treated

cells against untreated cells (control cells), for the non-irradiated (NI-SI) and

irradiated (I-SI) conditions, and the ratio of the stimulation indexes as determined as

the overall stimulation index (I-SI/NI-SI).

156

2.8. Statistical analysis

Results were expressed as mean ± standard error of least three independent

experiments. Statistical analysis were conducted using one-way analysis of

variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons and

by two-sample t-test using the Statistica software (v. 7.0; StatSoft. Inc., Tulsa, OK,

USA).

3. Results and discussion

3.1. 3T3 Neutral Red Uptake phototoxicity test

By using the 3T3 NRU phototoxic test, the cytotoxicity of the cells treated with

increasing concentrations of the compounds and irradiated with non-toxic dose of

UVA light was compared to non-irradiated cells. Fig. 1 illustrated the dose response

curves in absence and presence of UV light. The statistical analysis by ANOVA did

not show significant difference between the reduction in cell viability of irradiated and

non-irradiated cells (p > 0.05) for the free dronedarone, suggesting that it may not be

phototoxic. This effect was also observed for the inclusion complexes, indicating that

the complexation did not alter the phototoxic potential of dronedarone. An exception

was the minor phototoxic effect observed for the inclusion complex with HP-β-CD

prepared by colyophilization (Fig. 1c), evidenced by the significant difference (p <

0.05) between the cell viability of irradiated and non-irradiated cells for the lower

concentration tested, performed with Dunnett’s multiple comparison test (non-

irradiated as control). The colyophilization technique involves the use of ethanol in

157

the sample preparation, and even though rotary evaporation removes it, residual

solvent would remain in the sample and could contribute to the cytotoxic effect.

Firstly, the radiation sensitivity of the cells to the light source was tested

following exposure to increasing doses of irradiation (1.0, 1.7, 1.9, 2.5 and 5.0 J/cm²),

using CPZ as positive control in the range of 0.35 to 90 µg/mL. Cell viability was

determined after 24 h using Neutral Red uptake. The quality requirement is cell

viability higher than 80% for non-irradiated control cells. In assessing the doses of

irradiation, the dose of 5.0 J/cm² had led to cell death; the dose of 2.5 J/cm² had

reduced cell viability to only about 42.4% (S.D. = 5.3); for the dose of 1.9 J/cm²

viability was about 64.4% (S.D. = 15.9) and dose of 1.7 J/cm² resulted in almost

100% cell viability. Thus, the 1.7 J/cm² dose was chose, as it met the quality criteria.

The dose of 1.0 J/cm² did not provide enough radiation to activate the phototoxic

potential of the compounds.

Noteworthy is that amiodarone can be classified as phototoxic in the in vitro

3T3 NRU phototoxic test, based on the minimum mean photo effect value (0.27),

which is higher than the value predicting phototoxicity (0.15). Amiodarone also

presents a high absorption in UV range, with the absorbance maxima at 242 nm and

a shoulder around 300 nm [26]. Amiodarone and its metabolite desethylamiodarone

are highly cytotoxic compounds and the main mechanism underlying cell damage is

the generation of active metabolites due to radiation. An example of such metabolites

is the reactive oxygen species, which may cause the destruction of DNA, cell

membranes as well as oxygenation of lipids [30]. The induced phototoxicity of

amiodarone is a response mainly to the UVA light range. In vivo experiments

suggested that amiodarone is accumulated in a higher concentration in dermis and,

as UVA can penetrate deeply in this layer of the skin and UVB only reaches the

158

epidermal basal cell layer, UVA is the radiation most responsible for the phototoxicity

[21,31]. The phototoxic reaction could persist for several months, as amiodarone has

a long half-life (40-55 days) [31,32]. Amiodarone molecule has the presence of iodine

on the aromatic ring, leading the molecule to be more lipophilic, increasing

accumulation in tissues where toxicity is known to occur: thyroid, lungs, liver, cornea,

skin and peripheral nerves [33].

On the other hand, the removal of iodine and the addition of the methane-

sulphonyl group in dronedarone molecule reduced the lipophilicity and, consequently,

its half-life (to approximately 24 h) and accumulation in tissue [1]. These molecular

alterations, performed with the intention to diminish the deleterious effects of

amiodarone, had a direct impact on drug lipophilicity and, consequently, on its

toxicity, which could thus explain the lack of phototoxicity found for dronedarone in

the 3T3 NRU phototoxicity assay.

159

Fig. 1. Dose response curves of DRO (A) and inclusion complexes prepared by

colyophilization with β-CD (B) and HP-β-CD (C), by lyophilization with β-CD (D) and

HP-β-CD (E) and by kneading following spray-drying with β-CD (F) and HP-β-CD (G)

in non-irradiated (diamonds) and irradiated (squares) in NIH-3T3 cells. Results are

presented as mean ± SE of three independent experiments, and statistical analysis

was performed with Dunnett’s multiple comparison test (*p < 0.05).

0

20

40

60

80

100

120

0 0,3 0,6 1,3 2,5 5,0 7,5 10,0 15,0

Via

bili

ty (

%)

Concentration (µg/mL)

-Irr

+Irr

0

20

40

60

80

100

120

0 0,3 0,6 1,25 2,5 5 7,5 10 15

Via

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0 0,3 0,6 1,25 2,5 5 7,5 10 15

Via

bili

ty (

%)


-Irr

+Irr

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Via

bili

ty (

%)


-Irr

+Irr

0

20

40

60

80

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Via

bili

ty (

%)


-Irr

+Irr

0

20

40

60

80

100

120

0 0,3 0,6 1,25 2,5 5 7,5 10 15

Via

bili

ty (

%)


-Irr

+Irr

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20

40

60

80

100

120

0 0,3 0,6 1,25 2,5 5 7,5 10 15

Via

bili

ty (

%)


-Irr

+Irr

A

FE

DC

B

G

*

160

3.2. Determination of the photosensitizing potential using THP-1 cells

In order to determine the effect of the compounds resulting in 75% viability

(CV75) 24 h after treatment, the concentration range 0.625-2.50 µg/mL of free drug

and inclusion complexes was tested in non-irradiated and irradiated conditions. Cell

viability was assessed by MTT reduction, calculated by the percentage of MTT

reduction by viable cells against the untreated control cells in irradiated and non-

irradiated conditions. Results are presented in Fig. 2, and for free dronedarone and

the inclusion complexes, CV75 was 1.25 µg/mL, since the concentration of 2.50

µg/mL presented cell viability lower than 75% and the concentration of 0.625 µg/mL

did not induce a significant response to UVA dose.

The effect of the compounds together with the UVA radiation on the release of

IL-8 from THP-1 cells was investigated, and the results for irradiated and non-

irradiated conditions are represented in Fig. 3. The free DRO and inclusion

complexes produced a dose-related increase of IL-8 release, which was statistically

significant under irradiated condition (p < 0.05). These findings suggested that free

dronedarone and its inclusion complexes with β-CD and HP-β-CD are able to

stimulate IL-8 release after irradiation, with the exception of the complex with β-CD

prepared by kneading following spray-drying.

In the irradiated condition, the IL-8 release from cells treated with inclusion

complexes at the concentration of 1.25 µg/mL was compared to that from the cells

treated with free DRO, by using the two-sample t-test. Results are shown in Fig. 4,

which displayed a significantly lower IL-8 release for the inclusion complex with β-CD

prepared by kneading following spray-drying (p < 0.05).

The sensitizing potential of DRO and the inclusion complexes was

161

investigated by using a methodology previously developed [35]. For non-irradiated

condition, all the compounds failed to induce IL-8 release, suggesting that they were

not sensitizers. In contrast, in the case of the inclusion complex with β-CD produced

by colyophilization (Fig. 3b) and the complex with HP-β-CD produced by kneading

and spray-dryer (Fig. 3g), a dose-related response was also observed for the non-

irradiated condition.

Considering that sensitization and photosensitization share common

mechanisms, and in order to confirm that the reaction occurred in response to UVA,

stimulation indexes (SI) were calculated, according to Martínez et al. [24]. The

calculation of SI was based on the release of IL-8 from cells treated with the

concentration of 1.25 µg/mL, the CV75. Indeed, it was calculated by the ratio of the

treated cells against untreated cells, for the non-irradiated (NI-SI) and irradiated (I-

SI), and the ratio of the stimulation indexes determined as the overall stimulation

index (I-SI/NI-SI). Figure 5 shows the respective stimulation indexes for the free drug

and the inclusion complexes.

162

Fig. 2. Cytotoxicity rates measure by the MTT assay for non-irradiated (gray) and irradiated (black) conditions. The concentration tested for dronedarone (D) and inclusion complexes (LH, RH, SH, SB, RB, LB) were 2.5 µg/mL (a), 1.25 µg/mL (b) and 0.625 µg/mL (c).

0

10

20

30

40

50

60

70

80

90

100

D LH RH SH SB RB LB

Ce

ll vi

abili

ty (

%)

2.5 µg/mL -Irr

2.5 µg/mL +Irr

0

10

20

30

40

50

60

70

80

90

100

D LH RH SH SB RB LB

Ce

ll vi

abili

ty (

%)

1.25 µg/mL -Irr

1.25 µg/mL +Irr

0

10

20

30

40

50

60

70

80

90

100

D LH RH SH SB RB LB

Ce

ll vi

abiit

y (%

)

0.625 µg/mL -Irr

0.625 µg/mL +Irr

a

c

b

163

Fig. 3 – IL-8 release induced by increasing concentrations of free DRO (a), inclusion complexes prepared by colyophilization with β-CD (b) and HP-β-CD (c), by lyophilization with β-CD (d) and HP-β-CD (e), and by kneading following spray-drying with β-CD (f) and HP-β-CD (g), and chlorpromazine (h) in non-irradiated (open circles) and irradiated cells (black squares). SI calculated for each concentration tested is also shown (black triangles). Results are presented as mean ± S.E.M., and statistical analysis was performed with two-sample t-test. (*p < 0.05; **p<0.01).

0

0,5

1

1,5

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2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

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SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


a*

*

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

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100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

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IL-8

(p

g/m

L)


b**

**

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


c**

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


d

*

**

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


e**

**

**

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


f

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,6 1,25 2,5

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


g**

*

*

**

** ** **

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0,00

50,00

100,00

150,00

200,00

250,00

300,00

350,00

400,00

0 0,1 0,3 0,7 1,5 3,0

SI (N

I-S

I/I-

SI)

IL-8

(p

g/m

L)


h

164

Fig. 4. Effects of DRO and inclusion complexes on IL-8 release. THP-1 cells (irradiated and non-irradiated) were treated with the compounds at a concentration of 1.25 µg/mL for 24 h. IL-8 release was measured by ELISA in culture supernatants, results expressed in pg/mL, representing the mean ± S.E.M. Statistical analysis was performed with two-sample t-test, with *p< 0.05 versus DRO.

Fig. 5 – The increases of IL-8 release expressed as stimulation indexes for non-irradiated (NI-SI) and irradiated cells (I-SI). An overall stimulation index (I-SI/NI-SI) was calculated as the ratio of the stimulation indexes in irradiated and non-irradiated cells. The concentrations assayed were: chlorpromazine (CPZ) 0.1 µg/mL, DRO 1.25 µg/mL and inclusion complexes (RB, RH, LB, LH, SB and SH) equivalent to 1.25 µg/mL of DRO.

0

50

100

150

200

250

300

350

400

IL-8

(pg/

mL)

Irr-

Irr+

*

0

0,5

1

1,5

2

2,5

3

3,5

CPZ DRO RB RH LB LH SB SH

Re

lati

ve s

tim

ula

tio

n i

nd

ex

NI-SI

I-SI

I-SI/NI-SI

165

Chlorpromazine, an antipsychotic drug with potential to induce photo irritating

and photoallergic reactions [20], was used as positive control in the assay, whose

overall stimulation index was 1.9. The result of the overall SI for the free DRO was

2.4, suggesting that DRO is a photosensitizer, with a SI value higher than the positive

control. The overall SI for all the inclusion complexes presented lower values in

comparison to the positive control, except the inclusion complex with β-CD prepared

by lyophilization, which presented a higher value. In contrast, the complex with β-CD

prepared by kneading following spray-drying presented the lowest overall SI value,

followed by the complex with HP-β-CD prepared by the same technique. Therefore, it

can be evidenced that they may induce the photoallergic reaction in a lower extend in

comparison to the free dronedarone.

Prior conducting the photoassay, the appropriate UVA dose was investigated,

considering the dose that do not induce a decrease on cell viability or a significant

release of IL-8 from supernatant of non-treated cells. Initially, a dose of 2.5 J/cm² was

tested; however, this exposure caused a reduction of 20 % on the cell viability on

control cells. The UVA dose of 1.9 J/cm² was tested and provided enough energy to

photoactivation without decreasing control cell viability, and was thus selected for

further experiments.

Photoallergic reactions are type IV hypersensitive delay responses mediated

by specific T-cells, requiring specific sensitization to a photo activated drug. The

photochemical reaction results in the formation of a complete antigen, involving

covalent drug-protein binding [31]. The mechanism of photocontact dermatitis is the

same of allergic contact dermatitis, which starts with Langerhans cells, a type of

dendritic cells resident in the skin, presenting the antigen and then migrating from the

epidermis to the dermis. In the lymph node, the Langerhans cells differentiate into

166

mature immunostimulatory cells by up-regulating the expression of several co-

stimulatory molecules such as CD2, CD11a, CD54 and CD58, and secreting various

cytokines such as IL-1beta and IL-8 [24,25,34].

Considering this mechanism occurring in vivo, alternative methods were

developed in order to investigate skin sensitization, by using dendritic and human

myeloid cell lines. The human monocytic leukemia cell line THP-1 has been

proposed as a model to identify sensitizers, considering that they respond by

elevating expression of co-stimulatory molecules, such as CD54 and CD56, as well

as the production of IL-8. IL-8 attracts immature dendrit cells and neutrophils, which

in response can release chemotactic mediators attracting T-cells [34]. Based on this

mechanism, a photoassay using the THP-1 cell line and the IL-8 release was

developed as an in vitro model to identify if the compounds are photoallergens [24].

Here, this photoassay was performed in order to determine the photosensitizing

potential of free DRO and its inclusion complexes with β-CD and HP-β-CD, in order

to investigate if the complexation with CDs could alter the photosensitization induced

by the free drug, as reported in a patient treated with dronedarone one month before

the appearance of photosensitivity [8].

Thus, considering our experimental data, mainly the overall SI values, we

suggested that the inclusion complexes with β-CD and HP-β-CD obtained by

kneading and spray-dryer could reduce the photosensitizing potential of DRO.

3.3. Cytotoxicity in HepG2 cells

In order to study the hepatotoxicity of free DRO and its inclusion complexes

with CD in vitro, the human hepatoma cell line HepG2 was exposed to increasing

167

concentrations of each sample. Fig. 6 displayed the cytotoxic effects after treatment

with free drug for 24 h. Concentrations in the range from 0.31 to 5.0 µg/mL did not

significantly decrease cell viability; however, the concentrations of 7.5, 10.0 and 15.0

µg/mL reduced the cell viability significantly (p < 0.0001). The concentration-

dependent decrease response was evidenced by the significant difference (p< 0.05)

between 7.5 µg/mL and the higher concentrations.

In a previous study [28], which investigated the mechanisms underlying in vitro

hepatotoxicity of dronedarone after exposure of HepG2 cells for 24 h, the drug

started to impaired mitochondrial function around 10 µM (5.932 µg/mL) and

cytotoxicity was observed at 20 µM (11.864 µg/mL). Our data is in line with those

results reported in this study, since at the concentration of 5.0 µg/mL a reduction of

20% on cell viability was observed, even though was not statistically significant from

the control cells, might indicating the beginning of mitochondrial toxicity.

Fig. 6 –Concentration response curve from 24 h-exposure of HepG2 cells to free DRO. Data are expressed as mean ± S.E.M. of three independent experiments performed in triplicate. Statistical analysis was performed using two-sample t-test. *p<0.05 versus control, **p<0.001 versus control, ***p<0.00001 versus control, p<0.05 versus 7.50 µg/mL.

0

10

20

30

40

50

60

70

80

90

100

0 0,31 0,63 1,25 2,50 5,00 7,50 10,00 15,00

MTT

re

du

ctio

n (%

co

ntr

ol)


*

**

***

168

The IC50-values of free DRO and the inclusion complexes are shown in Fig. 7.

The overall results suggested that the MTT assay provided a higher IC50 value for

the free drug in comparison to the inclusion complexes. The lower IC50 value was

found for the inclusion complex with β-CD prepared by kneading following spray-

drying, followed by the complex with HP-β-CD prepared by the same technique.

However, when analyzed by one-way ANOVA following Dunnett’s multiple

comparison test, using the IC50 value of the free DRO as control, the inclusion

complexes IC50-values did not show significant differences, suggesting that the

complexation with CDs did not alter DRO hepatotoxicity significantly.

Fig. 7 – Cytotoxicity of free DRO and inclusion complexes prepared by colyophilization with β-CD (RB) and HP-β-CD (RH), by lyophilization with β-CD (LB) and HP-β-CD (LH) and by kneading following spray-drying with β-CD (SB) and HP-β-CD (SH) expressed as IC50 values (µg/mL) in HepG2 cells measured by the MTT assay. Data represent the mean ± S.E.M. of three independent experiments.

4. Conclusions

In this study, the phototoxic, photosensitizing and hepatotoxic potentials of

0

1

2

3

4

5

6

7

8

DRO RB RH LB LH SB SH

IC5

0 (

µg

//m

L)

169

free DRO and its inclusion complexes with β-CD e HP-β-CD were investigated by

using in vitro cell-based models. The results of the 3T3 NRU phototoxicity assay

showed that free DRO did not present phototoxic effects. In the photosensitization

studies using THP-1 cell and IL-8, the free drug showed the potential to induce skin

sensitization, as induced IL-8 release after irradiation. The free drug and inclusion

complexes were also tested concerning their hepatotoxicity by using HepG2 cells

and the results found for DRO were similar to a previous study. Overall, the inclusion

complexes with β-CD and HP-β-CD prepared by lyophilization did not alter the

phototoxic, photosensitizing or hepatotoxic potentials of DRO, while the inclusion

complex with HP-β-CD prepared by colyophilization presented a minor phototoxic

effect. The inclusion complexes prepared by kneading following spray-dryer

technique showed to induce a lower IL-8 release after irradiation in comparison to the

pure drug. Finally, these findings could promote the development of a new

pharmaceutical dosage form with reduced side effects.

Conflict of Interest Statement: The authors declare that they have no conflict of

interest.

Acknowledgements: This work was supported by the Brazilian National Council for

Scientific and Technological Development (CNPq) [grant numbers 401069/2014-1

and 447548/2014-0]; FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio

Grande do Sul) [grant number 2293-2551/14-0]; and CAPES (Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior).

170

References

[1] S.H. Hohnloser, H.J.G.M. Crijns, M. van Eickels, C. Gaudin, R.L. Page, C. Torp-Pedersen, S.J. Connolly, N. Engl. J. Med. 360 (2009) 668.

[2] S.J. Connolly, A.J. Camm, J.L. Halperin, C. Joyner, M. Alings, J. Amerena, D. Atar, Á. Avezum, P. Blomström, M. Borggrefe, A. Budaj, S.-A. Chen, C.K. Ching, P. Commerford, A. Dans, J.-M. Davy, E. Delacrétaz, G. Di Pasquale, R. Diaz, P. Dorian, G. Flaker, S. Golitsyn, A. Gonzalez-Hermosillo, C.B. Granger, H. Heidbüchel, J. Kautzner, J.S. Kim, F. Lanas, B.S. Lewis, J.L. Merino, C. Morillo, J. Murin, C. Narasimhan, E. Paolasso, A. Parkhomenko, N.S. Peters, K.-H. Sim, M.K. Stiles, S. Tanomsup, L. Toivonen, J. Tomcsányi, C. Torp-Pedersen, H.-F. Tse, P. Vardas, D. Vinereanu, D. Xavier, J. Zhu, J.-R. Zhu, L. Baret-Cormel, E. Weinling, C. Staiger, S. Yusuf, S. Chrolavicius, R. Afzal, S.H. Hohnloser, N. Engl. J. Med. 365 (2011) 2268.

[3] C. Patel, G.-X. Yan, P.R. Kowey, Circulation 120 (2009) 636. [4] S.D. Han, S.W. Jung, S.W. Jang, H.J. Jung, M. Son, B.M. Kim, M.J. Kang,

Chem. Pharm. Bull. 63 (2015) 295. [5] T.G.A. Australian Government, Department of Health and Ageing, (2010) 1. [6] U.S. Food and Drug Administration, (2014). [7] C.-W. Siu, M.P. Wong, C.-M. Ho, C.-L. Lam, H.-F. Tse, Arch Intern Med 170

(2012) 516. [8] S. Kuo, K. Menon, R. V. Kundu, Cutis 94 (2014) E10. [9] A. Popielec, T. Loftsson, Int. J. Pharm. (2017). [10] S.S. Jambhekar, P. Breen, Drug Discov. Today 21 (2016) 356. [11] Z. Fülöp, P. Saokham, T. Loftsson, Int. J. Pharm. 472 (2014) 282. [12] T. Loftsson, S.B. Vogensen, M.E. Brewster, F. Konráðsdóttir, J. Pharm. Sci. 96

(2007) 2532. [13] T. Loftsson, D. Duchêne, Int. J. Pharm. 329 (2007) 1. [14] S. V. Kurkov, T. Loftsson, Int. J. Pharm. 453 (2013) 167. [15] E.M.M. Del Valle, Process Biochem. 39 (2004) 1033. [16] C. Fernandes, I. Encarnacao, A. Gaspar, J. Garrido, F. Borges, E.M. Garrido,

Int. J. Photoenergy 2014 (2014). [17] V. Aiassa, A. Zoppi, M.C. Becerra, I. Albesa, M.R. Longhi, Carbohydr. Polym.

152 (2016) 672. [18] E.M.H. Yee, J.M. Hook, M.M. Bhadbhade, O. Vittorio, R.P. Kuchel, M.B.

Brandl, R.D. Tilley, D.S. Black, N. Kumar, Carbohydr. Polym. 165 (2017) 444. [19] P.S.S. Lima, A.M. Lucchese, H.G. Araújo-Filho, P.P. Menezes, A.A.S. Araújo,

L.J. Quintans-Júnior, J.S.S. Quintans, Carbohydr. Polym. 151 (2016) 965. [20] N.J. Neumann, A. Blotz, G. Wasinska-Kempka, M. Rosenbruch, P. Lehmann,

H.J. Ahr, H.W. Vohr, J. Photochem. Photobiol. B Biol. 79 (2005) 25. [21] T. Baccarin, M. Mitjans, D. Ramos, E. Lemos-Senna, M.P. Vinardell, J.

Photochem. Photobiol. B Biol. 153 (2015) 127. [22] S. Onoue, K. Kawamura, N. Igarashi, Y. Zhou, M. Fujikawa, H. Yamada, Y.

Tsuda, Y. Seto, S. Yamada, J. Pharm. Biomed. Anal. 47 (2008) 967. [23] M.H. Kleinman, TrAC - Trends Anal. Chem. 49 (2013) 100. [24] V. Martínez, V. Galbiati, E. Corsini, R. Martín-Venegas, M.P. Vinardell, M.

Mitjans, Toxicol. Vitr. 27 (2013) 1920. [25] M. Mitjans, V. Galbiati, L. Lucchi, B. Viviani, M. Marinovich, C.L. Galli, E.

Corsini, Toxicol. Vitr. 24 (2010) 1803.

171

[26] I. Consideration, (2004) 1. [27] M.P. Vinardell, Regul. Toxicol. Pharmacol. 71 (2015) 198. [28] A. Felser, K. Blum, P.W. Lindinger, J. Bouitbir, S. Krähenbühl, Toxicol. Sci. 131

(2013) 480. [29] T. Mosmann, J. Immunol. Methods 65 (1983) 55. [30] K. Jaworski, I. Walecka, L. Rudnicka, M. Gnatowski, D.A. Kosior, Med. Sci.

Monit. 20 (2014) 2369. [31] R. Mang, H. Stege, J. Krutmann, in:, Contact Dermatitis, Springer Berlin

Heidelberg, Berlin, Heidelberg, 2011, pp. 155–163. [32] J.W.M. Cheng, Am. J. Heal. Pharm. 67 (2010) S26. [33] J.C. Laughlin, P.R. Kowey, J. Cardiovasc. Electrophysiol. 19 (2008) 1220. [34] M. Mitjans, B. Viviani, L. Lucchi, C.L. Galli, M. Marinovich, E. Corsini, Toxicol.

Vitr. 22 (2008) 386. [35] D. Corti, V. Galbiati, N. Gatti, M. Marinovich, C.L. Galli, E. Corsini, Toxicol. Vitr.

29 (2015) 1339. [36] T. Schilirò, L. Alessandria, S. Bonetta, E. Carraro, G. Gilli, Chemosphere 145

(2016) 89.

172

5 DISCUSSÃO

174

5 DISCUSSÃO

A dronedarona é um novo fármaco antiarrítmico análogo a amiodarona,

aprovado para a manutenção do ritmo cardíaco normal em pacientes com fibrilação

atrial e, assim, indicado para reduzir os riscos de hospitalização (HOHNLOSER et

al., 2009). É um fármaco pertencente à classe II do Sistema de Classificação

Biofarmacêutica, por possuir baixa solubilidade em água (0,64 mg/mL), sendo esta

também dependente do pH (1-2 mg/mL em pH 3-5; < 0,01 mg/mL em fluido gástrico

e intestinal). Além disso, após administração oral, sofre com a interação fármaco-

alimento, além de intenso metabolismo de primeira passagem, levando a uma

biodisponibilidade absoluta de apenas 15% (AUSTRALIAN GOVERNMENT,

DEPARTMENT OF HEALTH AND AGEING, 2010; HAN et al., 2015a, 2015b). As

ciclodextrinas são oligossacarídeos cíclicos utilizados como adjuvantes

farmacêuticos com a finalidade de aumentar a solubilidade, estabilidade físico-

química e a biodisponibilidade de fármacos. Sua estrutura molecular possui uma

conformação tronco-cônica, conferindo um caráter externo hidrofílico e uma

cavidade interna lipofílica, que tem a capacidade de encapsular moléculas

hidrofóbicas no seu interior, através de interações não-covalentes (JAMBHEKAR;

BREEN, 2016; LOFTSSON; DUCHÊNE, 2007).

A cromatografia líquida de alta eficiência é um método amplamente utilizado

na para a quantificação de fármacos em formulações, estudos de estabilidade e para

a determinação das constantes de estabilidade dos complexos de inclusão (MURA,

2014). Dessa maneira, validou-se procedimento para determinação de dronedarona

em complexos de inclusão com ciclodextrinas e em comprimidos comerciais

conforme demonstrado no artigo 1. O método consistiu no uso de coluna C18 e fase

móvel composta por solução tampão de ácido acético glacial a 0.3% (pH 4,9) e

acetonitrila na proporção 35:65 (v/v), com vazão de 1,0 mL/min. Em relação a

validação do método, o método apresentou-se linear na faixa de 5 a 100 µg/mL (r =

0,9999; y = 44111,42 x + 21073,45); preciso, com valores de DPR para

repetibilidade e precisão intermediária inferiores ao preconizado (BRASIL, 2003), e

exato (média das recuperações > 99%). A robustez foi demonstrada por pequenas

alterações nos parâmetros individuais e por delineamento fatorial fracionário, através

do qual demonstrou-se que nenhum dos fatores ou a combinação destes exerceu

efeito significativo na determinação do teor de fármaco. Na avaliação da

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especificidade, realizada através dos estudos de degradação forçada com o fármaco

puro, com os comprimidos comerciais e complexo de inclusão com HP-β-CD,

evidenciaram a susceptibilidade do fármaco à hidrólise alcalina com aquecimento,

seguindo cinética de primeira ordem. Após degradação ácida, um pico adicional foi

detectado em 4,8 min. Em relação a fotólise, observou-se a presença de um pico

adicional no cromatograma do fármaco livre e de dois picos no cromatograma da

solução de comprimidos. Entretanto, nenhum pico adicional foi detectado na solução

do complexo de inclusão. Além disso, a degradação foi 9 vezes menor no complexo

de inclusão em comparação às soluções do fármaco livre e dos comprimidos

comerciais, sugerindo um efeito foto-protetor da CD, semelhante ao descrito em

outros estudos (FERNANDES et al., 2014; POPIELEC; LOFTSSON, 2017).

O desenvolvimento, a caracterização e a avaliação da citotoxicidade dos

complexos de inclusão de dronedarona com β-CD e HP-β-CD foram apresentados

no artigo 2. Os complexos foram preparados por três diferentes técnicas: liofilização,

coliofilização e malaxagem seguida de secagem por aspersão. Durante o

desenvolvimento dos complexos de inclusão, verificou-se a eficiência de

solubilização do fármaco na presença de 10 mM de CD foi de três vezes. Na

avaliação do teor dos complexos, também se verificou que uma proporção molar de

1 mol de fármaco para 10 mols de CDs forneceu teores acima de 85% para a

maioria dos complexos e, por isso, essa foi a razão molar escolhida para o preparo

das formulações. Através do estudo das constantes de complexação, obtido pelo

cálculo da constante de estabilidade, verificou-se que a HP-β-CD é um melhor

solubilizante do que a β-CD, que pode ser devido a HP-β-CD possuir maior

solubilidade do que a CD natural (BREWSTER; LOFTSSON, 2007).

O fármaco e os complexos de inclusão foram caracterizados por calorimetria

exploratória diferencial (DSC), difração de raios-X de pó (DRXP), espectroscopia no

infravermelho (IV) e microscopia eletrônica de varredura (MEV). Nos estudos de

caracterização por DSC, pode-se avaliar que a dronedarona apresenta ponto de

fusão de 144°C, que associado aos resultados obtidos por DRXP, mostraram alto

grau de cristalinidade do fármaco. Esses achados foram confirmados pela imagem

obtida por MEV, na qual o fármaco é caracterizado por formas retangulares. A β-CD

também apresentou padrão cristalino nos estudos de DRXP, e nas imagens de MEV

mostrou-se na forma de grandes partículas poliédricas. Já a HP-β-CD apresentou

um halo amorfo característico no difratograma e na análise da morfologia por MEV

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foram observadas partículas esféricas.

Em relação à caracterização dos complexos com β-CD preparados por

liofilização (LB) e coliofilização (RB) por DSC, DRXP e IV, os resultados demonstram

que ambos os complexos foram muito semelhantes, com uma grande redução na

cristalinidade após a complexação, evidenciada pela DRXP. No DSC houve

mudança dos pontos de fusão, resultado da mudança na estrutura cristalina. As

fotomicrografias também evidenciaram morfologias semelhantes, com a formação de

blocos irregulares. Por outro lado, os complexos preparados com HP-β-CD pelas

duas técnicas (LH e RH) mostraram um maior grau de amorfização, indicado pela

presença do halo amorfo semelhante ao da HP-β-CD isolada. Nos resultados de

DSC, os pontos de fusão foram alterados para temperaturas superiores e houve

redução da entalpia de fusão. Os complexos obtidos por malaxagem seguida de

secagem por aspersão com β-CD (SB) e HP-β-CD (SH) também apresentam

resultados semelhantes, com o completo desaparecimento dos picos intensos da

dronedarona e da β-CD (no caso do complexo SB) nos respectivos difratogramas e

pela alteração e desaparecimento dos pontos de fusão nos complexo SH e SB,

respectivamente, nas curvas de DSC. Essas alterações são atribuídas à perda da

estrutura cristalina causada pelo encapsulamento. As fotomicrografias de ambos os

complexos mostraram a formação de partículas esféricas de tamanho reduzido. Nos

espectros de infravermelho obtidos para todos os complexos, a banda característica

do fármaco em 1334 cm -1 desapareceu, confirmando os resultados obtidos pelas

demais técnicas e sugerindo a formação dos complexos de inclusão.

Como resultado da complexação, foi observado um aumento de cerca de 4

vezes na solubilidade aquosa da dronedarona. Nos estudos de dissolução em fluido

gástrico simulado (pH 1,2) e em tampão fosfato pH 6,8, a dronedarona livre

apresentou baixa porcentagem dissolvida mesmo após 2 h de ensaio. Já os

complexos de inclusão aumentaram a taxa de dissolução do fármaco em cerca de

4,5 vezes em pH 1,2 e em 9 vezes em pH 6,8, sugerindo uma melhora na dissolução

do fármaco no trato gastrintestinal.

Dentre os métodos in vitro para a avaliação da toxicidade de produtos e

substâncias químicas, as técnicas que utilizam células vivas são as mais

empregadas, pois mantém a intrínseca complexidade celular. A citotoxicidade das

soluções do produto comercial submetidas à degradação forçada foi avaliada em

fibroblastos, conforme descrito no artigo 1, no qual se detectou potencial citotóxico

177

nas amostras submetidas à fotólise. A citotoxicidade dos complexos de inclusão

também foi investigada na mesma linhagem celular, como apresentado no artigo 2,

no qual se demonstrou uma redução significativa na citotoxicidade do fármaco para

os complexos de inclusão preparados por coliofilização (RB e RH), malaxagem

seguida de secagem por aspersão (SB e SH) e para o complexo obtido por

liofilização com β-CD (LB). Além disso, considerando-se os resultados obtidos neste

estudo e alguns efeitos adversos da dronedarona relatados na literatura como

reação de fotossensibilidade (KUO; MENON; KUNDU, 2014) e hepatotoxicidade

(FDA, 2014), buscou-se investigar o efeito da complexação com ciclodextrinas sobre

a fototoxicidade, hepatotoxicidade e potencial fotossensibilizante do fármaco,

apresentados no artigo 3. O potencial fototóxico da dronedarona livre e dos

complexos de inclusão foi determinado pelo teste de fototoxicidade in vitro 3T3 NRU,

um método alternativo validado. Os resultados obtidos no ensaio não indicaram

fototoxicidade, com exceção do complexo com HP-β-CD preparado por coliofilização

(RH), que apresentou leve efeito fototóxico. A fotossensibilização foi avaliada

utilizando a linhagem celular de leucemia monocítica aguda humana (THP-1) como

modelo e a liberação de interleucina-8 como biomarcador. O fármaco livre e os

complexos de inclusão induziram a liberação de IL-8 após a irradiação. Entretanto, o

complexo de inclusão com β-CD preparado por secagem por aspersão (SB) foi

capaz de estimular a liberação de IL-8 em níveis mais baixos, em comparação ao

fármaco livre, sugerindo uma redução do potencial fotosensibilizante do fármaco. O

complexo com HP-β-CD preparado por secagem por aspersão (SH) também

apresentou índice de estimulação da produção de IL-8 inferior ao fármaco livre. Nos

estudos de hepatotoxicidade com células de tumorais de hepatoma humano

(HepG2), os complexos obtidos por secagem por aspersão (SB e SH) apresentaram

os menores valores de concentração inibitória 50% (IC50), entretanto não houve

redução significativa da citotoxicidade quando comparadas a IC50 da dronedarona

livre.

Em relação as concentrações plasmáticas medidas em pacientes após a

administração repetida de 400 mg duas vezes ao dia juntamente com a refeição, de

acordo com o declarado pelo fabricante, a Cmax da dronedarona foi de 84-147

ng/mL (PATEL; YAN; KOWEY, 2009; U.S. FOOD AND DRUG ADMINISTRATION,

2014). Considerando-se as concentrações de fármaco analisadas nos ensaios de

citotoxicidade in vitro, verificou-se que as amostras fotodegradadas demonstraram

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potencial fototóxico em concentrações acima de 1,0 µg mL-1 (artigo 1); o fármaco

puro reduziu a viabilidade celular em concentrações acima de 5,0 µg mL-1 e a

complexação com ambas ciclodextrinas reduziu o potencial citotóxico da

dronedarona (artigo 2) e a mesma concentração de 5,0 µg mL-1 reduziu a

viabilidade celular em células HepG2 (artigo 3). Assim, as concentrações de

fármaco nas quais houve redução da viabilidade celular, nos diferentes ensaios, são

muito superiores às concentrações detectadas in vivo após administração oral,

sugerindo segurança após uso do produto comercial. Entretanto, se for considerada

outra via de administração (ex: intravenosa), os resultados dos ensaios podem ser

relevantes para a escolha da dose administrada, permitindo a avaliação do

desenvolvimento da toxicidade, como uma etapa preliminar aos estudos in vivo.

A partir dos resultados obtidos, conclui-se que a formação de complexos de

inclusão aumentou significativamente a solubilidade aquosa e a taxa de dissolução

do fármaco em pHs fisiológicos. Além disso, os resultados preliminares de

citotoxicidade in vitro demonstraram uma redução do potencial citotóxico da

dronedarona após a complexação. Ainda que sejam as etapas iniciais do

desenvolvimento, os complexos de inclusão β-CD e HP-β-CD com podem apoiar o

desenvolvimento de novos sistemas de liberação contendo dronedarona, visando a

melhoria da eficácia e segurança do tratamento da fibrilação atrial.

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6 CONCLUSÃO

182

6 CONCLUSÃO

- Complexos de inclusão de cloridrato de dronedarona com β-CD e HP-β-CD

foram preparados com êxito utilizando três diferentes técnicas: liofilização,

coliofilização e malaxagem seguida de secagem por aspersão;

- O método por cromatografia a líquido de alta eficiência foi validado e

utilizado para a determinação de dronedarona em comprimidos e em complexos de

inclusão com ciclodextrinas;

- A caracterização dos complexos de inclusão foi realizada por calorimetria

exploratória diferencial (DSC), difração de raios-X de pó (DRXP), espectroscopia de

infravermelho (IV) e microscopia eletrônica de varredura (MEV), comprovando a

formação de complexos de inclusão verdadeiros;

- A complexação com ciclodextrinas aumentou a solubilidade do fármaco em

aproximadamente 4 vezes. Além disso, os percentuais de dissolução em pH 1,2 e

6,8 tiveram aumento de aproximadamente 4,5 e 9 vezes, respectivamente;

- A citotoxicidade dos complexos de inclusão preparados por coliofilização

(RB e RH) e malaxagem seguida de secagem por aspersão (SB e SH), bem como

do complexo obtido por liofilização com β-CD (LB), foi significativamente inferior à do

fármaco livre;

- A dronedarona não apresentou potencial fototóxico, entretanto evidenciou-se

seu potencial fotossensibilizante. Neste contexto, demonstrou-se que os complexos

obtidos por malaxagem seguida de secagem por aspersão poderiam reduzir o

potencial fotossensibilizante do fármaco.

- Todos os complexos apresentaram melhoria nas características físico-

químicas, quando comparados ao fármaco puro. Entretanto, destacam-se os

complexos obtidos por malaxagem seguida de secagem por aspersão, pela redução

da citotoxicidade. Assim, a formulação é promissora para o desenvolvimento de um

produto farmacêutico com propriedade farmacêutica potencializada e com efeitos

183

adversos reduzidos.

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REFERÊNCIAS

AGUIAR, U. N. et al. Preparação e caracterização do complexo de inclusão do oléo essencial de Croton zehntneri com betaciclodextrina. Química Nova, v. 37, n. 1, p. 50–55, 2014. AHIRRAO, V. K. et al. Stress degradation studies of dronedarone in pharmaceutical dosage form by a validated stability-indicating LC method. Journal of the Chilean Chemical Society, v. 57, n. 3, p. 1272–1276, 2012. ALLEN, L. V.; POPOVICH, N. G.; ANSEL, H. C. Formas farmacêuticas e sistemas de liberação de fármacos. Porto Alegre: Artmed, 2007. 776 p. AUSTRALIAN GOVERNMENT, DEPARTMENT OF HEALTH AND AGEING, T. G. A. Australian Public Assessment Report for Dronedarone Hydrochloride. Disponível em: <https://www.tga.gov.au/sites/default/files/auspar-multaq.pdf>. Acesso em: 6 jan. 2017. BERRIDGE, M.; TAN, A. S. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcelullar localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Archives of Biochemistry and Biophysics, v. 303, n. 2, p. 474–482, 1993. BHATT, K. K.; PATELIA, E. M.; AMIN, I. Development of a validated stability-indicating RP-HPLC method for dronedarone hydrochloride in pharmaceutical formulation. Journal of Analytical & Bioanalytical Techniques, v. 4, n. 1, p. 1–6, 2013. BIN JARDAN, Y. A.; GABR, R. Q.; BROCKS, D. R. Pharmacokinetics of dronedarone in rat using a newly developed high-performance liquid chromatographic assay method. Biomedical Chromatography, v. 28, n. 8, p. 1070–1074, 2014. BIN JARDAN, Y. A.; BROCKS, D. R. The pharmacokinetics of dronedarone in normolipidemic and hyperlipidemic rats. Biopharmaceutics & Drug Disposition, v. 37, p. 345–351, 2016. BOLDERMAN, R.W.; HERMANS, J. J. R.; MAESSEN, J. G. Determination of the class III antiarrhythmic drugs dronedarone and amiodarone, and their principal metabolites in plasma and myocardium by high-performance liquid chromatography and UV-detection. Journal of Chromatography B, v. 877, p. 1727-1731, 2009. BRASIL - Agência Nacional de Vigilância Sanitária. Resolução RE n° 899: Guia para validação de métodos analíticos e bioanalíticos, 2003. BRASIL - Agência Nacional de Vigilância Sanitária. Resolução RE n°4.920, de 5 de novembro de 2009. BREWSTER, M. E.; LOFTSSON, T. Cyclodextrins as pharmaceutical solubilizers. Advanced Drug Delivery Reviews, v. 59, n. 7, p. 645–666, 2007.

185

BRUNTON, L.L., LAZO, J.S, PAR ER, .L. Goodman & Gilman’s As bases farmacológicas da terapêutica. 11 ed. Rio de Janeiro: McGraw-Hill, 2006. CHADHA, R.; BALI, A.; BANSAL, G. Identification and characterization of stress degradation products of dronedarone hydrochloride employing LC-UV/PDA, LC–MS/TOF and MSn studies. Journal of Pharmaceutical and Biomedical Analysis, v. 118, p. 139–148, 2016. CHENG, J. W. M. New and emerging antiarrhythmic and anticoagulant agents for atrial fibrillation. American Journal of Health-System Pharmacy, v. 67, n. 9_Supplement_5, p. S26–S34, 2010. CHUGH, S. S. et al. Worldwide epidemiology of atrial fibrillation: a global burden of disease 2010 study. Circulation, v. 129, n. 8, p. 837–847, 2014. DA CUNHA FILHO, M. S. S.; SÁ-BARRETO, L. C. L. Utilização de ciclodextrinas na formação de complexos de inclusão de interesse farmacêutico. Revista de Ciências Farmacêuticas Básica e Aplicada, v. 28, n. 1, p. 1–9, 2007. EMA – European Medicines Agency. European Public Assesment Report. Multaq: EPAR – Summary for the public, London, UK, 2012. Disponível em < http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/001043/WC500044534.pdf >. Acesso em: 4 agosto. 2017. FELSER, A. et al. Mechanisms of hepatocellular toxicity associated with dronedarone - A comparison to amiodarone. Toxicological Sciences, v. 131, n. 2, p. 480–490, 2013. FERNANDES, C. et al. Influence of Hydroxypropyl-beta-Cyclodextrin on the Photostability of Fungicide Pyrimethanil. International Journal of Photoenergy, v. 2014, 2014. FUSTER, V. et al. ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation-executive summary: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines and the European Society of Cardiology Commi. European Heart Journal, v. 27, n. 16, p. 1979–2030, 2006. GAUTIER, P. et al. Electrophysiologic characterization of dronedarone in guinea pig ventricular cells. Journal of Cardiovascular Pharmacology, v. 41, n. 2, p. 191–202, 2003. GIORDANO, F.; NOVAK, C.; MOYANO, J. R. Thermal analysis of cyclodextrins and their inclusion compounds. Thermochimica Acta, v. 380, n. 2, p. 123–151, 2001. GO, A.; HYLEK, E.; PHILLIPS, K. Prevalence of diagnosed atrial fibrillation in adults. JAMA, v. 285, n. 18, p. 2370–2375., 2001. HAN, S. D. et al. Preparation of Solid Dispersion of Dronedarone Hydrochloride with

186

Soluplus ® by Hot Melt Extrusion Technique for Enhanced Drug Release. Chemical and Pharmaceutical Bulletin, v. 63, n. 4, p. 295–299, 2015a. HAN, S. D. et al. Reduced Food-Effect on Intestinal Absorption of Dronedarone by Self-microemulsifying Drug Delivery System (SMEDDS). Biological & Pharmaceutical Bulletin, v. 38, n. 7, p. 1026–1032, 2015b. HIGUCHI, T.; CONNORS, K. A. Phase solubility techniques. In: REILLY, C. N. (Ed.). Advances in Analytical Chemistry and Instrumentation. New York, N.Y.: Interscience Publishers, 1965. v. 4p. 117–212. HOHNLOSER, S. H. et al. Effect of dronedarone on cardiovascular events in atrial fibrillation. The New England Journal of Medicine, v. 360, p. 668–678, 2009. HOY, S. M.; KEAM, S. J. Dronedarone. Drugs, v. 69, n. 12, p. 1647–1663, 2009. IACOVINO, R. et. al. Cyclodextrins as complexant agents: preparation and applications. Current Organic Chemistry, v. 21, p. 162-176, 2017. JAMBHEKAR, S. S.; BREEN, P. Cyclodextrins in pharmaceutical formulations I: Structure and physicochemical properties, formation of complexes, and types of complex. Drug Discovery Today, v. 21, n. 2, p. 356–362, 2016. JOUYBAN, A. Handbook of solubillity data for pharmaceuticals.Boca Raton: CRC Press, 2010, 538 p. KIM, K.; PARK, H.; LIM, K.-M. Phototoxicity: Its Mechanism and Animal Alternative Test Methods. Toxicological Research, v. 31, n. 2, p. 97–104, 2015. KIM, M.; BAEK, I. Effect of dronedarone on the pharmacokinetics of carvedilol following oral administration to rats. European Journal of Pharmaceutical Sciences, v. 111, p. 13–19, 2018. KUO, S.; MENON, K.; KUNDU, R. V. Photosensitivity reaction from dronedarone for atrial fibrillation. Cutis, v. 94, p. E10–E11, 2014. KURKOV, S. V.; LOFTSSON, T. Cyclodextrins. International Journal of Pharmaceutics, v. 453, n. 1, p. 167–180, 2013. LALEVÉE, N. et al. Effects of amiodarone and dronedarone on voltage-dependent sodium current in human cardiomyocytes. Journal of Cardiovascular Electrophysiology, v. 14, n. 8, p. 885–890, 2003. LANDGE, S. B. et al. Stability indicating RP-HPLC method for the determination of dronedarone hydrochloride and its potential process-related impurities in bulk drug and pharmaceutical dosage form. American Journal of Analytical Chemistry, v. 4, n. 6, p. 323–335, 2013. LAUGHLIN, J. C.; KOWEY, P. R. Dronedarone: A new treatment for atrial fibrillation. Journal of Cardiovascular Electrophysiology, v. 19, n. 11, p. 1220–1226, 2008.

187

LOFTSSON, T. Cyclodextrins and the Biopharmaceutics Classification System of Drugs. Journal of Inclusion Phenomena and Macrocyclic Chemistry, v. 44, p.63–67, 2002. LOFTSSON, T., BREWSTER, M.E. Cyclodextrins as functional excipients: methods to enhance complexation efficiency. Journal of Pharmaceutical Sciences, v.101, n. 9, p. 3019–3032, 2012. LOFTSSON, T.; DUCHÊNE, D. Cyclodextrins and their pharmaceutical applications. International Journal of Pharmaceutics, v. 329, n. 1–2, p. 1–11, 2007. LOFTSSON, T.; HREINSDÓTTIR, D.; MÁSSON, M. Evaluation of cyclodextrin solubilization of drugs. International Journal of Pharmaceutics, v. 302, n. 1–2, p. 18–28, 2005. LOFTSSON, T.; HREINSDÓTTIR, D.; MÁSSON, M. The complexation efficiency. Journal of Inclusion Phenomena and Macrocyclic Chemistry, v. 57, n. 1–4, p. 545–552, 2007. LOFTSSON, T.; MÁSSON, M.; BREWSTER, M. E. Self-association of cyclodextrins and cyclodextrin complexes. Journal of Pharmaceutical Sciences, v. 93, n. 5, p. 1091–1099, 2004. LOFTSSON, T., MOYA-ORTEGA, M. D.; ALVAREZ-LORENZO, C.; CONCHEIRO A. Pharmacokinetics of cyclodextrins and drugs after oral and parenteral administration of drug/cyclodextrin complexes. Journal of Pharmacy and Pharmacology, v. 68, n. 5, p. 544–555, 2016. LOFTSSON, T., MUELLERTZ, A., SIEPMANN, J. For the Special IJP issue “Poorly Soluble Drugs.” International Journal of Pharmaceutics, v. 453, n. 1, p. 1–2, 2013. LYNCH, A. M.; WILCOX, P. Experimental and Toxicologic Pathology Review of the performance of the 3T3 NRU in vitro phototoxicity assay in the pharmaceutical industry. Experimental and Toxicologic Pathology, v. 63, n. 3, p. 209–214, 2011. LYRA, M. A M. et al. Ferramentas analíticas aplicadas à caracterizaçã o de complexos de inclusão fármaco-ciclodextrina. Revista de Ciências Farmacêuticas Básica e Aplicada, v. 31, n. 2, p. 117–124, 2010. MARCOLINO, A. I. P. et al. Determination of dronedarone in the pharmaceutical dosage form by a stability-indicating micellar electrokinetic chromatography method. Analytical Methods, v. 5, n. 12, p. 3106, 2013. MARTÍNEZ, V. et al. Establishment of an in vitro photoassay using THP-1 cells and IL-8 to discriminate photoirritants from photoallergens. Toxicology in Vitro, v. 27, n. 6, p. 1920–7, 2013. MCKIM, J. M. J. Building a Tiered Approach to In Vitro Predictive Toxicity Screening  A Focus on Assays with In Vivo Relevance. Combinatorial Chemistry and High Throughput Screening, v. 13, p. 188–206, 2010.

188

MIRANDA, J.C. et al. Cyclodextrins and ternary complexes: technology to improve solubility of poorly soluble drugs. Brazilian Journal of Pharmaceutical Sciences, v. 47, n. 4, p. 665-681, 2011. MITJANS, M. et al. Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THP-1 cells. Toxicology in Vitro, v. 22, p. 386-395, 2008. MITJANS, M.et al. Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro. Toxicology in Vitro, v. 24, p. 1803-1809, 2010. MOSMANN, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, v. 65, n. 1–2, p. 55–63, 16 dez. 1983. MURA, P. Analytical techniques for characterization of cyclodextrin complexes in aqueous solution  A review. Journal of Pharmaceutical and Biomedical Analysis, v. 101, p. 238–250, 2014. MURA, P. Analytical techniques for characterization of cyclodextrin complexes in the solid state: A review. Journal of Pharmaceutical and Biomedical Analysis, v. 113, p. 226–238, set. 2015. NATTEL, S. Therapeutic implications of atrial fibrillation mechanisms: Can mechanistic insights be used to improve AF management? Cardiovascular Research, v. 54, n. 2, p. 347–360, 2002. NEUMANN, N. J. et al. Evaluation of phototoxic and photoallergic potentials of 13 compounds by different in vitro and in vivo methods. Journal of Photochemistry and Photobiology B: Biology, v. 79, n. 1, p. 25–34, 2005. NOGUEIRA, D. R. et al. Comparative sensitivity of tumor and non-tumor cell lines as a reliable approach for in vitro cytotoxicity screening of lysine-based surfactants with potential pharmaceutical applications. International Journal of Pharmaceutics, v. 420, n. 1, p. 51–58, 2011. OECD. In Vitro 3T3 NRU phototoxicity test. OECD Guideline for Testing of Chemicals, n. April, p. 1–15, 2004. OLIVEIRA, R.; SANTOS, D.; COELHO, P. Ciclodextrinas: formação de complexos e sua aplicação farmacêutica. Revista da Faculdade de Ciências da Saúde, v. 6, p. 70-83, 2009. ONOUE, S. et al. Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: Classification criteria and application to drug candidates. Journal of Pharmaceutical and Biomedical Analysis, v. 47, n. 4–5, p. 967–972, 2008. PAGE, R. L.; HAMAD, B.; KIRKPATRICK, P. Dronedarone. Nature Reviews Drug Discovery, v. 8, n. 10, p. 769–770, 2009.

189

PATEL, C.; YAN, G.-X.; KOWEY, P. R. Dronedarone. Circulation, v. 120, n. 7, p. 636–644, 2009. POPIELEC, A.; LOFTSSON, T. Effects of cyclodextrins on the chemical stability of drugs. International Journal of Pharmaceutics, in press. PYDIMARRY, S. P. R.; CHOLLETI, V. K.; VANGALA, R. R. Stability-indicating UPLC method for determining related substances and degradants in dronedarone. Journal of Chromatographic Science, v. 52, n. 7, p. 666–675, 2014. ROLIM, L. A. et al. Aplicações de revestimento em formas farmacêuticas sólidas na indústria farmacêutica Coating applications of solid pharmaceutical forms in pharmaceutical industry. Revista Brasileira de Farmácia, v. 90, n. 3, p. 224–230, 2009. ROWE, R. C; SHESKEY, P. J.; OWEN, S. C. Handbook of Pharmaceutical Excipients. 5 ed. London: Phamaceutical Press, 2006. 918 p. SA BARRETO, C. L.; CUNHA-FILHO, M. S. S. Ciclodextrina: Importante excipiente farmacêutico funcional. Latin American Journal of Pharmacy, v. 27, n. 4, p. 629–636, 2008. SINGH, B. N.; ALIOT, E. Newer antiarrhythmic agents for maintaining sinus rhythm in atrial fibrillation  simplicity or complexity? v. 9, p. 17–25, 2007. SPIELMANN, H. Progress in establishing in vitro alternatives to animal experiments – Ethical and scientific challenges. AATEX, v. 11, n. 2, p. 89–100, 2005. SUN, W.; SARMA, J. S. M.; SINGH, B. N. Chronic and acute effects of dronedarone on the action potential of rabbit atrial muscle preparations: comparison with amiodarone. Journal of Cardiovascular Pharmacology, v. 39, n. 5, p. 677–684, 2002. TAKAHASHI, A. I. et al. A literature review of cyclodextrin inclusion complexes characterization - Part II: X-ray diffraction, infrared spectroscopy and nuclear magnetic resonance. International Journal of Pharmaceutical Sciences Review and Research, v. 12, n. 1, p. 8–15, 2012. TAKAHASHI, A. I.; VEIGA, F. J. B.; FERRAZ, H. G. A literature review of cyclodextrin inclusion complexes characterization - Part III  differential scanning calorimetry and thermogravimetry. International Journal Of Pharmaceutical Sciences Review and Research, v. 12, n. 1, p. 16–20, 2012. TONDEPU, N. et al. A stability indicating HPLC method for dronedarone in bulk drugs and pharmaceutical dosage forms. American Journal of Analytical Chemistry, v. 3, n. 8, p. 544–551, 2012. U.S. FOOD AND DRUG ADMINISTRATION. FDA labeling information: Revised Multaq label. Disponível em:

190

<http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/022425s025lbl.pdf>. Acesso em: 11 dez. 2016. UEKAMA, K. Design and evaluation of cyclodextrin-based drug formulation. Chemical and Pharmaceutical Bulletin, v. 52, n. 8, p. 900–915, 2004. VARRÓ, A et al. Electrophysiological effects of dronedarone (SR 33589), a noniodinated amiodarone derivative in the canine heart: comparison with amiodarone. British Journal of Pharmacology, v. 133, n. 5, p. 625–634, 2001. VEENA, M. C.; SENTHIL KUMAR, S. K.; PARTHIBAN, S. Preparation and evaluation of porous pellets with anti-arrhythmic drug for controlled release. Indian Journal of Pharmaceutical Science and Research, v. 3, n. 1, p. 20–31, 2013. WATANABE, Y., KIMURA, J. Acute inhibitory effect of dronedarone, a noniodinated benzofuran analogue of amiodarone, on Na+ / Ca2+ exchange current in guinea pig cardiac ventricular myocytes. Naunyn- Schmiedeberg’s Archives of Pharmacology, v. 337, p. 371-376, 2008. XIE, C. et al. Simultaneous determination of dronedarone and its active metabolite debutyldronedarone in human plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study. Journal of Chromatography B, v. 879, n. 28, p. 3071–3075, 2011. YALTA, K. et al. Dronedarone: a promising alternative for the management of atrial fibrillation. Cardiovascular Drugs and Therapy, v. 23, n. 5, p. 385–393, 2009. YEOLE, B. D. et al. Preparation of nanoparticles of poorly water soluble dronedarone by antisolvent addition technique using natural polymer as a stabilizer. Journal of Pharmaceutical Research and Clinical Practice, v. 6, n. 4, p. 8–16, 2016. ZANATTA, C. F. et al. Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines. Food and Chemical Toxicology, v. 48, n. 1, p. 70–5, 2010. ZONI-BERISSO, M. et al. Epidemiology of atrial fibrillation  European perspective. Clinical Epidemiology, v. 6, p. 213–220, 2014.

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